Antisense antiviral compounds and methods for treating a filovirus infection

ABSTRACT

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Filoviridae family, and in the treatment of a viral infection. The compounds and methods relate to the treatment of viral infections in mammals including primates by Ebola and Marburg viruses. The antisense antiviral compounds are morpholino oligonucleotides having: a) a nuclease resistant backbone, b) 15-40 nucleotide bases, and c) a targeting sequence of at least 15 bases in length that hybridizes to a target region selected from the following: i) the Ebola virus AUG start site region of VP24; ii) the Ebola virus AUG start site region of VP35; iii) the Marburg virus AUG start site region of VP24; or iv) the Marburg virus AUG start site region of NP.

This application is a continuation of U.S. patent application Ser. No.11/433,840, filed May 11, 2006 now U.S. Pat. No. 7,507,196, which is acontinuation-in-part of U.S. patent application Ser. No. 11/264,444,filed Oct. 31, 2005, now U.S. Pat. No. 7,524,829, which claims thebenefit of priority to U.S. provisional patent application Ser. No.60/671,694 filed Apr. 14, 2005, and U.S. provisional patent applicationSer. No. 60/624,277 filed Nov. 1, 2004. All applications areincorporated in their entirety herein by reference.

FIELD OF THE INVENTION

This invention relates to antisense oligonucleotide compounds for use intreating an infection by a virus of the Filoviridae family and antiviraltreatment methods employing the compounds. More specifically, it relatesto treatment methods and compounds for treating viral infections inmammals including primates by Ebola and Marburg viruses.

REFERENCES

-   Agrawal, S., S. H. Mayrand, et al. (1990). “Site-specific excision    from RNA by RNase H and mixed-phosphate-backbone    oligodeoxynucleotides.” Proc Natl Acad Sci USA 87(4): 1401-5.-   Arora, V. and P. L. Iversen (2001). “Redirection of drug metabolism    using antisense technology.” Curr Opin Mol Ther 3(3): 249-57.-   Blommers, M. J., U. Pieles, et al. (1994). “An approach to the    structure determination of nucleic acid analogues hybridized to RNA.    NMR studies of a duplex between 2′-OMe RNA and an oligonucleotide    containing a single amide backbone modification.” Nucleic Acids Res    22(20): 4187-94.-   Bonham, M. A., S. Brown, et al. (1995). “An assessment of the    antisense properties of RNase H-competent and steric-blocking    oligomers.” Nucleic Acids Res 23(7): 1197-203.-   Borio, L., T. Inglesby, et al. (2002). “Hemorrhagic fever viruses as    biological weapons: medical and public health management.” Jama    287(18): 2391-405.-   Boudvillain, M., M. Guerin, et al. (1997). “Transplatin-modified    oligo(2′-O-methyl ribonucleotide)s: a new tool for selective    modulation of gene expression.” Biochemistry 36(10): 2925-31.-   Bray, M., K. Davis, et al. (1998). “A mouse model for evaluation of    prophylaxis and therapy of Ebola hemorrhagic fever.” J Infect Dis    178(3): 651-61.-   Burnett, J., E. A. Henchal, et al. (2005). “The evolving field of    biodefence: Therapeutic developments and diagnostics.” Nat Rev Drug    Disc 4: 281-297.-   Connolly, B. M., K. E. Steele, et al. (1999). “Pathogenesis of    experimental Ebola virus infection in guinea pigs.” J Infect Dis 179    Suppl 1: S203-17.-   Cross, C. W., J. S. Rice, et al. (1997). “Solution structure of an    RNA×DNA hybrid duplex containing a 3′-thioformacetal linker and an    RNA A-tract.” Biochemistry 36(14): 4096-107.-   Dagle, J. M., J. L. Littig, et al. (2000). “Targeted elimination of    zygotic messages in Xenopus laevis embryos by modified    oligonucleotides possessing terminal cationic linkages.” Nucleic    Acids Res 28(10): 2153-7.-   Ding, D., S. M. Grayaznov, et al. (1996). “An    oligodeoxyribonucleotide N3′-->P5′ phosphoramidate duplex forms an    A-type helix in solution.” Nucleic Acids Res 24(2): 354-60.-   Egholm, M., O. Buchardt, et al. (1993). “PNA hybridizes to    complementary oligonucleotides obeying the Watson-Crick    hydrogen-bonding rules.” Nature 365(6446): 566-8.-   Feldmann, H., S. Jones, et al. (2003). “Ebola virus: from discovery    to vaccine.” Nat Rev Immunol 3(8): 677-85.-   Feldmann, H. and M. P. Kiley (1999). “Classification, structure, and    replication of filoviruses.” Curr Top Microbiol Immunol 235: 1-21.-   Feldmann, H., H. D. Klenk, et al. (1993). “Molecular biology and    evolution of filoviruses.” Arch Virol Suppl 7: 81-100.-   Felgner, P. L., T. R. Gadek, et al. (1987). “Lipofection: a highly    efficient, lipid-mediated DNA-transfection procedure.” Proc Natl    Acad Sci USA 84(21): 7413-7.-   Gait, M. J., A. S. Jones, et al. (1974). “Synthetic-analogues of    polynucleotides XII. Synthesis of thymidine derivatives containing    an oxyacetamido- or an oxyformamido-linkage instead of a    phosphodiester group.” J Chem Soc [Perkin 1] 0(14): 1684-6.-   Gee, J. E., I. Robbins, et al. (1998). “Assessment of high-affinity    hybridization, RNase H cleavage, and covalent linkage in translation    arrest by antisense oligonucleotides.” Antisense Nucleic Acid Drug    Dev 8(2): 103-11.-   Geisbert, T. W. and L. E. Hensley (2004). “Ebola virus: new insights    into disease aetiopathology and possible therapeutic interventions.”    Expert Rev Mol Med 6(20): 1-24.-   Geisbert, T. W., L. E. Hensley, et al. (2003). “Treatment of Ebola    virus infection with a recombinant inhibitor of factor VIIa/tissue    factor: a study in rhesus monkeys.” Lancet 362(9400): 1953-8.-   Jahrling, P. B., T. W. Geisbert, et al. (1999). “Evaluation of    immune globulin and recombinant interferon-alpha2b for treatment of    experimental Ebola virus infections.” J Infect Dis 179 Suppl 1:    S224-34.-   Lesnikowski, Z. J., M. Jaworska, et al. (1990). “Octa(thymidine    methanephosphonates) of partially defined stereochemistry: synthesis    and effect of chirality at phosphorus on binding to    pentadecadeoxyriboadenylic acid.” Nucleic Acids Res 18(8): 2109-15.-   Mertes, M. P. and E. A. Coats (1969). “Synthesis of carbonate    analogs of dinucleosides. 3′-Thymidinyl 5′-thymidinyl carbonate,    3′-thymidinyl 5′-(5-fluoro-2′-deoxyuridinyl) carbonate, and    3′-(5-fluoro-2′-deoxyuridinyl) 5′-thymidinyl carbonate.” J Med Chem    12(1): 154-7.-   Moulton, H. M., M. H. Nelson, et al. (2004). “Cellular uptake of    antisense morpholino oligomers conjugated to arginine-rich    peptides.” Bioconjug Chem 15(2): 290-9.-   Nelson, M. H., D. A. Stein, et al. (2005). “Arginine-rich peptide    conjugation to morpholino oligomers: effects on antisense activity    and specificity.” Bioconjug Chem 16(4): 959-66.-   Peters, C. J. and J. W. LeDuc (1999). “An introduction to Ebola: the    virus and the disease.” J Infect Dis 179 Suppl 1: ix-xvi.-   Sanchez, A., M. P. Kiley, et al. (1993). “Sequence analysis of the    Ebola virus genome: organization, genetic elements, and comparison    with the genome of Marburg virus.” Virus Res 29(3): 215-40.-   Strauss, J. H. and E. G. Strauss (2002). Viruses and Human Disease.    San Diego, Academic Press.-   Summerton, J. and D. Weller (1997). “Morpholino antisense oligomers:    design, preparation, and properties.” Antisense Nucleic Acid Drug    Dev 7(3): 187-95.-   Toulme, J. J., R. L. Tinevez, et al. (1996). “Targeting RNA    structures by antisense oligonucleotides.” Biochimie 78(7): 663-73.-   Warfield, K. L., J. G. Perkins, et al. (2004). “Role of natural    killer cells in innate protection against lethal ebola virus    infection.” J Exp Med 200(2): 169-79.

BACKGROUND OF THE INVENTION

Minus-strand (−) RNA viruses are major causes of human suffering thatcause epidemics of serious human illness. In humans the diseases causedby these viruses include influenza (Orthomyxoviridae), mumps, measles,upper and lower respiratory tract disease (Paramyxoviridae), rabies(Rhabdoviridae), hemorrhagic fever (Filoviridae, Bunyaviridae andArenaviridae), encephalitis (Bunyaviridae) and neurological illness(Bornaviridae). Virtually the entire human population is thought to beinfected by many of these viruses (e.g. respiratory syncytial virus)(Strauss and Strauss 2002).

The order Mononegavirales is composed of four minus strand RNA virusfamilies, the Rhabdoviridae, the Paramyxoviridae, the Filoviridae andthe Bornaviridae. The viruses in these families contain a single strandof non-segmented negative-sense RNA and are responsible for a wide rangeof significant diseases in fish, plants, and animals. Viruses withsegmented (−) RNA genomes belong to the Arenaviridae, Bunyaviridae andOrthomyxoviridae families and possess genomes with two, three and sevenor eight segments, respectively.

The expression of the five to ten genes encoded by the members of theMononegavirales is controlled at the level of transcription by the orderof the genes on the genome relative to the single 3′ promoter. Geneorder throughout the Mononegavirales is highly conserved. Genes encodingproducts required in stoichiometric amounts for replication are alwaysat or near the 3′ end of the genome while those whose products areneeded in catalytic amounts are more promoter distal (Strauss andStrauss 2002). The segmented (−) RNA viruses encode genes with similarfunctions to those encoded by the Mononegavirales. Other features ofvirion structure and replication pathways are also shared among the (−)RNA viruses.

For some (−) RNA viruses, effective vaccines are available (e.g.influenza, mumps and measles virus) whereas for others there are noeffective vaccines (e.g. Ebola virus and Marburg virus). In general, noeffective antiviral therapies are available to treat an infection by anyof these viruses. As with many other human viral pathogens, availabletreatment involves supportive measures such as anti-pyretics to controlfever, fluids, antibiotics for secondary bacterial infections andrespiratory support as necessary.

The development of a successful therapeutic for filoviruses Ebola andMarburg virus is a long-sought and seemingly difficult endeavor(Geisbert and Hensley 2004). Although they cause only a few hundreddeaths worldwide each year, filoviruses are considered a significantworld health threat and have many of the characteristics commonlyassociated with biological weapons since they can be grown in largequantities, can be fairly stable, are highly infectious as an aerosol,and are exceptionally deadly (Borio, Inglesby et al. 2002). Filovirusesare relatively simple viruses of 19 Kb genomes and consist of sevengenes which encode nucleoprotein (NP), glycoprotein (GP), four smallerviral proteins (VP24, VP30, VP35 and VP40), and the RNA-dependent RNApolymerase (L protein) all in a single strand of negative-sensed RNA(Feldmann and Kiley 1999). The development of an effective therapeuticfor Ebola virus has been hindered by a lack of reagents and a clearunderstanding of filovirus pathogenesis, disparity between animalmodels, and both the difficulty and danger of working with Ebola virusin biosafety level (BSL)-4 conditions (Geisbert and Hensley 2004;Burnett, Henchal et al. 2005). Administration of type I interferons,therapeutic vaccines, immune globulins, ribavirin, and other nucleosideanalogues have been somewhat successful in rodent Ebola virus models,but not in infected nonhuman primates (Jahrling, Geisbert et al. 1999;Geisbert and Hensley 2004; Warfield, Perkins et al. 2004). Ebola virusfrequently causes severe disseminated intravascular coagulation andadministration of a recombinant clotting inhibitor has recently shown toprotect 33% of rhesus monkeys (Geisbert, Hensley et al. 2003; Geisbertand Hensley 2004). Host-directed therapeutics alone have not proven tobe a sufficiently efficacious therapeutic approach. A well-orchestratedsequence-specific attack on viral gene expression is required for ahighly successful anti-filovirus therapeutic and treatment regimen.

In view of the severity of the diseases caused by (−) RNA viruses, inparticular members of the Filoviridae family of viruses, and the lack ofeffective prevention or therapies, it is therefore an object of thepresent invention to provide therapeutic compounds and methods fortreating a host infected with a (−) RNA virus.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, an anti-viral antisense compoundeffective in inhibiting replication within a host cell of an Ebola virusor Marburg virus. The has

a) a nuclease-resistant backbone,

b) 15-40 nucleotide bases,

c) a targeting sequence that is complementary to a target sequencecomposed of at least 12 contiguous bases within an AUG start-site regionof a positive-strand mRNA identified by one of the Filovirus mRNAsequences selected from the group consisting of SEQ ID NOS: 67-70, 71,72-75, and 76; and

(d) the ability to form a heteroduplex structure with the viral targetregion, wherein said heteroduplex structure is (i) composed of thepositive sense strand of the virus and the oligonucleotide compound, and(ii) characterized by a Tm of dissociation of at least 45° C.

For treating an Ebola virus infection, the compound may have a targetingsequence that is complementary to a target sequence composed of at least12 contiguous bases within the VP35 AUG start-site region identified bya target sequence selected from the group consisting of SEQ IDNOS:67-70. Exemplary targeting sequences include those identified by SEQID NOS. 21-26.

In another embodiment for treating an Ebola virus infection, thecompound may have a targeting sequence that is complementary to a targetsequence composed of at least 12 contiguous bases within the VP24 AUGstart-site region identified by a target sequence selected from thegroup consisting of SEQ ID NOS:72-75. Exemplary targeting sequencesinclude SEQ ID NOS:34-41.

For treating a Marburg virus infection, the compound may have atargeting sequence that is complementary to a target sequence composedof at least 12 contiguous bases within the VP35 AUG start-site regionidentified by a target sequence identified by SEQ ID NO:71. An exemplarytargeting sequence is selected from the group consisting of SEQ IDNOS:47 and 48.

In another embodiment for treating a Marburg virus infection, thecompound may have a targeting sequence that is complementary to a targetsequence composed of at least 12 contiguous bases within the VP24 AUGstart-site region identified by a target sequence identified by SEQ IDNOs:76. An exemplary targeting sequence is identified by SEQ ID NOs:57.

The compound may be composed of morpholino subunits linked byphosphorous-containing intersubunit linkages that join a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.The morpholino subunits may be joined by phosphorodiamidate linkages inaccordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. Inan exemplary compound, X═NR₂, and where each R is independently hydrogenor methyl.

At least 2 and no more than half of the total number of intersubunitlinkages may be positively charged at physiological pH. In thisembodiment, the morpholino subunits may be joined by phosphorodiamidatelinkages, in accordance with the structure:

where Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.

The compound may be conjugated to an arginine-rich polypeptide thatenhances the uptake of the compound into host cells. Exemplaryarginine-rich polypeptides have one of the sequences selected from thegroup consisting of SEQ ID NOS:61-66.

Also disclosed is a method of treating an Ebola or Marburg Filovirusinfection in a subject, by administering to the subject, atherapeutically effective amount of an oligonucleotide compound of thetype described above.

In still another aspect, the invention includes a method of vaccinatinga mammalian subject against Ebola virus by pretreating the subject withthe above compound, and exposing the subject to the Ebola virus,preferably in an attenuated form.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the repeating subunit segment of several preferredmorpholino oligonucleotides, designated A through D, constructed usingsubunits having 5-atom (A), six-atom (B) and seven-atom (C-D) linkinggroups suitable for forming polymers.

FIGS. 2A-2G show the backbone structures of various oligonucleotideanalogs with uncharged backbones and FIG. 2H shows a preferred cationiclinkage structure.

FIGS. 3A-3C illustrate the components and morphology of a filovirus(3A), and show the arrangement of viral genes in the Ebola virus (Zaire)(3B), and the Marburg virus (3C).

FIGS. 4A-4B show the target regions (SEQ ID NO: 1 and SEQ ID NO:42) of 6antisense compounds (SEQ ID NOS: 21, 22, 23, 24, 25, and 26) targetedagainst the VP35 gene in Ebola virus.

FIG. 5 is a plot showing cytotoxicity in Vero cell culture, expressed asa percent control, as a function of antisense type and concentration.

FIGS. 6A-6C are photomicrographs of Vero cells in culture (6A) in theabsence of Ebola virus infection and antisense treatment; (6B) withEbola virus infection but no antisense treatment; and (6C) with Ebolavirus infection and treatment with VP35-AUG (SEQ ID NO: 17) antisensecompound.

FIG. 7 is a plot of treatment efficacy, expressed as a fraction of mousesurvivors 10 days post infection, as a function of VP35 antisenselength.

FIG. 8 is a plot of treatment efficacy, expressed as percent survival,as a function of dose of various combinations of antisense compounds.

FIG. 9A shows the schedule of the experimental protocol. FIG. 9B plotsthe fraction of mouse survivors with various dose schedules of antisensecompounds.

FIG. 10 is a schematic of the treatment schedule for a trial using PMOto treat Ebola infection in nonhuman primates.

FIG. 11A-G shows Ebola-specific PMOs protect mice from lethal Ebolavirus infection. (A) Survival of mice pretreated at 4 and 24 hoursbefore EBOV infection with 500 μg of PMOs targeting VP24 (♦), VP35 (▪),L (▴), or with an unrelated sequence (x). (B) Survival of micepretreated with 1 mg (⋄), 0.1 mg (□), or 0.01 mg (Δ) of a combination ofthe VP24, VP35, and L) PMOs or 1 mg (♦), 0.1 mg (▪), or 0.01 mg (▴) ofVP35 PMO only or an unrelated sequence (x). (C) Survival in mice treated24 hours following EBOV infection with 1 mg (♦), 0.1 mg (▪), or 0.01 mg(▴) of the combination of PMOs or an unrelated sequence (x). (D-G)C57Bl/6 mice were challenged intraperitoneally with 1000 plaque-formingunits of EBOV following treatment with PMOs. Immunoperoxidase stain isbrown with hematoxylin counterstain. Viral antigen within the spleen(100×) of a mouse treated with scrambled PMO (D) or the EBOV PMOs (E)three days after EBOV infection. Diffuse staining pattern in the livers(600×) of the scrambled PMO-treated mice (F) on day 6 of EBOV infection,compared to focal areas of infection in the mice treated with thecombination of PMOs (G).

FIG. 12 shows that treatment of guinea pigs with antisense PMOsincreases survival following lethal Ebola virus infection. Hartleyguinea pigs were treated intraperitoneally with 10 mg each of VP24,VP35, and L PMO in PBS at −24 (▴), +24 (●), or +96 (▪) hours postchallenge. Control guinea pigs were injected with PBS only (x). Theguinea pigs were infected subcutaneously with ˜1000 pfu of EBOV andmonitored for illness for 21 days. The data are presented as percentsurvival for each group (n=6).

FIG. 13 shows that Ebola-specific PMOs reduce viral replication in vivo.Viral titers in tissues from mice treated with a combination of PMO andinfected with 1000 pfu of EBOV. Samples of the liver, spleen, and kidneywere taken at 3 or 6 days post challenge (dpc), macerated, and analyzedfor viral titer using plaque assay. The data are presented as the meanviral titer of 3 mice with error bars representing the standarddeviation.

FIG. 14A-C shows the immune responses of PMO-treated mice followingsurvival of Ebola virus infection. (A) PMO-treated C57BL/6 mice thathave previously survived EBOV infection generate EBOV-specific CD8⁺responses. Pooled splenocytes from three PMO-treated EBOV survivors werere-stimulated in vitro with EBOV-specific VP35 or NP peptides, anirrelevant Lassa NP peptide as a negative control, or PMA/ionomycin as apositive control. The stimulated cells were stained after for 4 hours inculture with anti-CD44 FITC, anti-IFN-γ PE, and anti-CD8 Cy-Chrome. Thepercent of CD44⁺, IFN-γ⁺ cells among CD8⁺ lymphocytes is indicated inthe upper right quadrant of each plot. These data are representative ofthe Ebola CD8 specific epitopes observed after challenge. (B) Totalserum anti-Ebola virus antibodies were measured in surviving mice priorto or 4 weeks following treatment and challenge. PMO mice were treatedwith the combination of PMOs 24 and 4 hours before challenge and theirantibody responses are compared with mice treated with Ebola VLPs 24hours before EBOV infection. The results are depicted as the endpointtiters of the individual mice (circles). The horizontal line in eachcolumn represents the geometric mean titer of the group. (C) Mice thatpreviously survived EBOV challenge following PMO treatment werere-challenged with 1000 pfu of mouse-adapted Ebola virus 4 weeks afterthe initial challenge. Results are plotted as percent survival for thePMO-treated mice (black) and naïve control mice (n=10 per group).

FIG. 15A-D shows that treatment of rhesus macaques with antisense PMOsprovide protection against lethal Ebola virus infection. (A) Survivalfollowing infection with 1000 pfu of EBOV in monkeys treated with acombination of PMOs (▪) or untreated monkeys (◯). The arrows indicatethe monkeys that died at the time indicated. (B-D) Viral titers (B),platelet counts (C), or alkaline phosphatase levels (D) in the blood ofthe PMO-treated monkeys [0646 (♦), 1438 (▴), 1496 (x), 1510 (▪)] or anuntreated monkey (◯).

FIG. 16 shows the increased antisense activity of PMOs with cationiclinkages targeting the EBOV VP24 mRNA in a cell free translation assay.PMOs used were 537-AUG (SEQ ID NO:34), 164-AUG+(SEQ ID NO:40),165-5′-term (SEQ ID NO:39) and 166-5′-term+(SEQ ID NO:41).

FIG. 17 shows the synthetic steps to produce subunits used toproduce+PMO containing the (1-piperazino) phosphinylideneoxy cationiclinkage as shown in FIG. 2H.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessotherwise indicated:

The terms “oligonucleotide analog” refers to an oligonucleotide having(i) a modified backbone structure, e.g., a backbone other than thestandard phosphodiester linkage found in natural oligo- andpolynucleotides, and (ii) optionally, modified sugar moieties, e.g.,morpholino moieties rather than ribose or deoxyribose moieties. Theanalog supports bases capable of hydrogen bonding by Watson-Crick basepairing to standard polynucleotide bases, where the analog backbonepresents the bases in a manner to permit such hydrogen bonding in asequence-specific fashion between the oligonucleotide analog moleculeand bases in a standard polynucleotide (e.g., single-stranded RNA orsingle-stranded DNA). Preferred analogs are those having a substantiallyuncharged, phosphorus containing backbone.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 50-100%, are uncharged at physiological pH, andcontain a single phosphorous atom. The analog contains between 8 and 40subunits, typically about 8-25 subunits, and preferably about 12 to 25subunits. The analog may have exact sequence complementarity to thetarget sequence or near complementarity, as defined below.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

A “morpholino oligonucleotide analog” is an oligonucleotide analogcomposed of morpholino subunit structures of the form shown in FIGS.1A-1D, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. The purine or pyrimidinebase-pairing moiety is typically adenine, cytosine, guanine, uracil,thymine or inosine. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in U.S. Pat. Nos.5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and5,506,337, all of which are incorporated herein by reference.

The subunit and linkage shown in FIG. 1B are used for six-atomrepeating-unit backbones, as shown in FIG. 1B (where the six atomsinclude: a morpholino nitrogen, the connected phosphorus atom, the atom(usually oxygen) linking the phosphorus atom to the 5′ exocyclic carbon,the 5′ exocyclic carbon, and two carbon atoms of the next morpholinoring). In these structures, the atom Y₁ linking the 5′ exocyclicmorpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusis any stable group which does not interfere with base-specific hydrogenbonding. Preferred X groups include fluoro, alkyl, alkoxy, thioalkoxy,and alkyl amino, including cyclic amines, all of which can be variouslysubstituted, as long as base-specific bonding is not disrupted. Alkyl,alkoxy and thioalkoxy preferably include 1-6 carbon atoms. Alkyl aminopreferably refers to lower alkyl (C₁ to C₆) substitution, and cyclicamines are preferably 5- to 7-membered nitrogen heterocycles optionallycontaining 1-2 additional heteroatoms selected from oxygen, nitrogen,and sulfur. Z is sulfur or oxygen, and is preferably oxygen.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 2B,where X═NH2, NHR, or NR2 (where R is lower alkyl, preferably methyl),Y═O, and Z═O, and Pi and Pj are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide, as seen in FIG. 2G. Also preferred are morpholinooligomers where the phosphordiamidate linkages are uncharged linkages asshown in FIG. 2G interspersed with cationic linkages as shown in FIG. 2Hwhere, in FIG. 2B, X=1-piperazino. In another FIG. 2B embodiment,X=lower alkoxy, such as methoxy or ethoxy, Y═NH or NR, where R is loweralkyl, and Z═O.

The term “substituted”, particularly with respect to an alkyl, alkoxy,thioalkoxy, or alkylamino group, refers to replacement of a hydrogenatom on carbon with a heteroatom-containing substituent, such as, forexample, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino,imino, oxo (keto), nitro, cyano, or various acids or esters such ascarboxylic, sulfonic, or phosphonic. It may also refer to replacement ofa hydrogen atom on a heteroatom (such as an amine hydrogen) with analkyl, carbonyl or other carbon containing group.

As used herein, the term “target”, relative to the viral genomic RNA,refers to at least one of the following: 1) a 125 nucleotide region thatsurrounds the AUG start codon of a viral messenger RNA and/or; 2) theterminal 30 bases of the 3′ terminal end of the minus-strand viral RNA(e.g. virion RNA or vRNA) and/or; 3) the terminal 25 bases of viral mRNAtranscripts.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide analog is directed, that is, the sequence towhich the oligonucleotide analog will hybridize by Watson-Crick basepairing of a complementary sequence. As will be seen, the targetsequence may be a contiguous region of the viral positive-strand mRNA orthe minus-strand vRNA.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the analog compound may be complementaryto the target sequence. For example, in an analog having 20 bases, only12-14 may be targeting sequences. Typically, the targeting sequence isformed of contiguous bases in the analog, but may alternatively beformed of non-contiguous sequences that when placed together, e.g., fromopposite ends of the analog, constitute sequence that spans the targetsequence. For example, as will be seen, the target and targetingsequences are selected such that binding of the analog to a portion of a125 nucleotide region associated with the AUG start codon of thepositive-sense RNA strand (i.e., mRNA) of the virus acts to disrupttranslation of the viral gene and reduce viral replication.

The term “AUG start site region” includes a 125 nucleotide region inboth the 5′ and 3′ direction relative to the AUG start codon of viralmRNAs. The region includes about 25 nucleotides downstream (i.e., in a3′ direction) and 100 nucleotides upstream (i.e., in a 5′ direction) asexemplified by the targets sequences shown as SEQ ID NOs:1-6, 67-70, and72-75 for Ebola virus and SEQ ID NOs: 8-13, 71 and 76 for Marburg virus.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the present invention haveat most one mismatch with the target sequence out of 10 nucleotides, andpreferably at most one mismatch out of 20. Alternatively, the antisenseoligomers employed have at least 90% sequence homology, and preferablyat least 95% sequence homology, with the exemplary targeting sequencesas designated herein.

An oligonucleotide analog “specifically hybridizes” to a targetpolynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a T_(m) substantially greater than 45°C., preferably at least 50° C., and typically 60° C.-80° C. or higher.Such hybridization preferably corresponds to stringent hybridizationconditions. At a given ionic strength and pH, the T_(m) is thetemperature at which 50% of a target sequence hybridizes to acomplementary polynucleotide. Again, such hybridization may occur with“near” or “substantial” complementary of the antisense oligomer to thetarget sequence, as well as with exact complementarity.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

A “heteroduplex” refers to a duplex between an oligonucleotide analogand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAseH, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

A “base-specific intracellular binding event involving a target RNA”refers to the specific binding of an oligonucleotide analog to a targetRNA sequence inside a cell. The base specificity of such binding issequence specific. For example, a single-stranded polynucleotide canspecifically bind to a single-stranded polynucleotide that iscomplementary in sequence.

An “effective amount” of an antisense oligomer, targeted against aninfecting filovirus, is an amount effective to reduce the rate ofreplication of the infecting virus, and/or viral load, and/or symptomsassociated with the viral infection.

As used herein, the term “body fluid” encompasses a variety of sampletypes obtained from a subject including, urine, saliva, plasma, blood,spinal fluid, or other sample of biological origin, such as skin cellsor dermal debris, and may refer to cells or cell fragments suspendedtherein, or the liquid medium and its solutes.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Treatment” of an individual or a cell is any type of interventionprovided as a means to alter the natural course of the individual orcell. Treatment includes, but is not limited to, administration of e.g.,a pharmaceutical composition, and may be performed eitherprophylactically, or subsequent to the initiation of a pathologic eventor contact with an etiologic agent. The related term “improvedtherapeutic outcome” relative to a patient diagnosed as infected with aparticular virus, refers to a slowing or diminution in the growth ofvirus, or viral load, or detectable symptoms associated with infectionby that particular virus.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, the oligonucleotide analog preferably has a substantiallyuncharged backbone, as defined below. Alternatively, the antisensecompound may be formulated in a complexed form, such as an agent havingan anionic backbone complexed with cationic lipids or liposomes, whichcan be taken into cells by an endocytotic mechanism. The analog also maybe conjugated, e.g., at its 5′ or 3′ end, to an arginine-rich peptide,e.g., a portion of the HIV TAT protein, or polyarginine, to facilitatetransport into the target host cell as described (Moulton, Nelson et al.2004). The compound may also have one or more cationic linkages toenhance antisense activity and/or cellular uptake. A preferred cationiclinkage is shown in FIG. 1B where X=(1-piperazino). The term “filovirus”refers collectively to members of the Filoviridae family of singlestranded (−) RNA viruses including Ebola and Marburg viruses listed inTable 1 below.

II. Targeted Viruses

The present invention is based on the discovery that effectiveinhibition of single-stranded, negative-sense RNA ((−) RNA) viruses canbe achieved by exposing cells infected with the virus to antisenseoligonucleotide analog compounds (i) targeted against the AUG startcodon of the positive sense viral mRNAs or the 3′ termini of thenegative strand viral RNA, and (ii) having physical and pharmacokineticfeatures which allow effective interaction between the antisensecompound and the virus within host cells. In one aspect, the oligomerscan be used in treating a mammalian subject infected with the virus.

The invention targets RNA viruses having genomes that are: (i) singlestranded, (ii) negative polarity, and (iii) less than 20 kb. Thetargeted viruses also synthesize a RNA species with positive polarity,the positive-strand or sense RNA, as the requisite step in viral geneexpression. In particular, targeted viruses include those of theFiloviridae family referred to herein as filoviruses. Targeted virusesorganized by family, genus and species are listed in Table 1. Variousphysical, morphological, and biological characteristics of each of theFiloviridae family, and members therein, can be found, for example, inTextbook of Human Virology, R. Belshe, ed., 2^(nd) Edition, Mosby, 1991,in “Viruses and Human Disease” (Strauss and Strauss 2002) and at theUniversal Virus Database of the International Committee on Taxonomy ofViruses (world wide web ncbi.nlm.nih.gov/ICTVdb/index.htm). Some of thekey biological characteristics of each family are summarized belowfollowing Table 1.

TABLE 1 Targeted viruses of the invention organized by family and genusFamily Genus Virus Filoviridae Marburg-like Marburg virus (MARV)Ebola-like Zaire Ebola virus (ZEBOV) Sudan Ebola virus (SEBOV) RestonEbola virus (REBOV) Cote d'Ivoire Ebola (ICEBOV)

A. Filoviridae

The Filoviridae family is composed of two members, Ebola virus (EBOV)and Marburg virus (MARV). Four species of Ebola have been identified todate and are named by the location of where they were identifiedincluding Ebola Ivory Coast (ICEBOV), Ebola Zaire (ZEBOV), Ebola Sudan(SEBOV) and Ebola Reston (REBOV). Ebola Reston is the only knownfilovirus that does not cause severe human disease. The filovirusstructure is pleomorphic with shapes varying from long filaments toshorter contorted structures. The viral filaments measure up to 14,000nm in length and have uniform diameter of 80 nm. The virus filament isenvelope in a lipid membrane. The virion contains one, single-stranded,negative sense RNA. The sequences of the viruses may be obtained throughpublicly available gene databases.

The first filovirus was recognized in 1967 after laboratory workers inMarburg Germany developed hemorrhagic fever following studies involvinghandling tissues from green monkeys. The Marburg outbreak led to 31cases and seven deaths. The first Ebola virus was identified in 1976following outbreaks of Ebola hemorrhagic fever in northern Zaire (nowthe Democratic Republic of Congo) and southern Sudan. Eventually, twodistinct viral isolates were recognized. Ebola Zaire was lethal in 90%of the infected cases and Ebola Sudan was lethal in 50% of the cases. Alist of Ebola hemorrhagic fever cases is compiled for Ebola Zaire inTABLE 2.

TABLE 2 Chronological order of Ebola Zaire Outbreaks Date Location HumanCases Deaths 1976 Zaire 318 280 (88%)  1977 Zaire 1  1 (100%) 1994 Gabon49 29 (59%) 1995 Dem. Rep. Congo 315 255 (81%)  1996 Gabon 31 21 (68%)1996 Gabon 60 45 (75%) 1996 South Africa 2  1 (50%) 2001 Gabon and Congo122 96 (79%)

The summary of these outbreak data include 899 cases resulting in 728deaths or an overall 81% rate of lethality observed over a period of 25years. A single case of Ebola Ivory Coast was reported in 1994 and thatinfection was not lethal. Finally, 4 outbreaks of Ebola Sudan from 1976to 2001 produced 744 cases resulting in 398 deaths or an overall rate oflethality of 53%. These observations indicate Ebola Zaire is the virusof greatest concern both in apparent prevalence and lethality. Itappears Ebola is transmitted to humans from ongoing life cycles inanimals other than humans which make it a zoonotic virus. Ebola canreplicate in various rodents such as mice, guinea pigs and some speciesof bats. Some types of bats are native to areas where the virus is foundwhich suggests the bat may be the natural host and viral reservoir. Oncea human is infected, person-to-person transmission is the means forfurther infections. During recorded outbreaks, individuals that caredfor or worked closely with infected people were at high risk of becominginfected. Nosocomial transmission has also been an important factor inthe spread of viral infection during outbreaks. In the laboratorysetting, viral spread through small-particle aerosols has been clearlydemonstrated.

The incubation period for Ebola hemorrhagic fever ranges from 2 to 21days. The clinical symptoms include abrupt onset of fever, headache,joint and muscle aches, sore throat and weakness. These symptoms arethen followed by diarrhea, vomiting and stomach pain which do not helpin diagnosis of infection. Diagnosis is suspected when this group ofsymptoms is observed in an area where Ebola is known to be active.Patients who die usually have not developed a significant immuneresponse to the virus at the time of death. There are no knowntreatments for filovirus infections.

The filovirus virus genome is approximately 19,000 bases ofsingle-stranded RNA that is unsegmented and in the antisense (i.e.negative sense) orientation. The genome encodes 7 proteins frommonocistronic mRNAs complementary to the vRNA as shown in FIG. 3. Areview of filoviruses can be found in Fields Virology and (Strauss andStrauss 2002).

Ebola Virus

Ebola virus (EBOV), a member of the family Filoviridae and the orderMononegavirales, is an enveloped, nonsegmented negative-strand RNA virusand is one of the most lethal human and nonhuman primate pathogensrecognized to date. Four subtypes of Ebola virus have been identified,including Zaire (ZEBOV), Sudan (SEBOV), Ivory Coast (ICEBOV), and Reston(REBOV) (Sanchez, Kiley et al. 1993). Human infection with subtype Zairecauses a fulminating, febrile, hemorrhagic disease resulting inextensive mortality (Feldmann, Klenk et al. 1993; Peters and LeDuc 1999;Feldmann, Jones et al. 2003).

Ebola virus particles have a filamentous appearance, but their shape maybe branched, circular, U- or 6-shaped, or long and straight. Virionsshow a uniform diameter of approximately 80 nm, but vary greatly inlength. Ebola virus particles consist of seven structural proteins. Theglycoprotein (GP) of Ebola virus forms spikes of approximately 7 nm,which are spaced at 5- to 10-nm intervals on the virion surface.

Marburg Virus

Marburg virus (MARV) was first recognized in 1967, when an outbreak ofhemorrhagic fever in humans occurred in Germany and Yugoslavia, afterthe importation of infected monkeys from Uganda. Thirty-one cases ofMARV hemorrhagic fever were identified that resulted in seven deaths.The filamentous morphology of the virus was later recognized to becharacteristic, not only of additional MARV isolates, but also of EBOV.MARV and EBOV are now known to be distinctly different lineages in thefamily Filoviridae, within the viral order Mononegavirales (Strauss andStrauss 2002).

Few natural outbreaks of MARV disease have been recognized, and allproved self-limiting, with no more than two cycles of human-to-humantransmission. However, the actual risks posed by MARV to global healthcannot be assessed because factors which restrict the virus to itsunidentified ecological niche in eastern Africa, and those that limitits transmissibility, remain unknown. Concern about MARV is furtherheightened by its known stability and infectivity in aerosol form. Arecent (2005) epidemic in eastern Africa caused at least 200 deaths andfurther increases the concern about MARV.

B. Target Sequences

The filovirus structure is pleomorphic with shapes varying from longfilaments to shorter contorted structures. The viral filaments measureup to 14,000 nm in length and have uniform diameter of 80 nm. The virusfilament is envelope in a lipid membrane. The virion contains one,single-stranded, negative sense RNA. The filovirus virus genome isapproximately 19,000 bases of single-stranded RNA that is unsegmentedand in the antisense orientation. The genome encodes 7 proteins frommonocistronic mRNAs complementary to the vRNA. A diagram of arepresentative filovirus and its genome is provided in FIGS. 3A-3C(taken from Fields Virology).

The targets selected were positive-strand (sense) RNA sequences thatspan or are just downstream (within 25 bases) or upstream (within 100bases) of the AUG start codon of selected Ebola virus proteins or the 3′terminal 30 bases of the minus-strand viral RNA. Preferred proteintargets are the viral polymerase subunits VP35 and VP 24, although L,nucleoproteins NP and VP30, are also contemplated. Among these earlyproteins are favored, e.g., VP35 is favored over the later expressed Lpolymerase. As will be seen, a preferred single-compound target is VP35that spans the AUG start site, and/or targets a region within 100 basesupstream or 25 bases downstream of the translational start site.Preferred combinations of targets include the VP35-AUG target plus theVP24-AUG start site (or the 100-base region upstream or 25-base-regiondownstream of the start site). The sequences for the VP35 AUG start-siteregion in the four Ebola strains are identified herein as SEQ IDNOS:67-70. The sequences for the VP24 AUG start-site region in the fourEbola strains are identified herein as SEQ ID NOS:72-75. The sequencesfor the VP35 AUG start-site region in the Marburg virus is identifiedherein as SEQ ID NO:71. The sequences for the VP24 AUG start-site regionin the Marburg virus is identified herein as SEQ ID NO:76. As will beseen below, a preferred targeting sequence is one that is complementaryto at least 12 contiguous bases in the one of the above sequencescorresponding to the start-site region of VP35 or VP24 of one of thefour Ebola virus subtypes or Marburg virus.

For the Ebola virus, exemplary target sequences against the VP35 regioninclude SEQ ID NOS: 21-26 for the Ebola Zaire strain and analogoustargeting sequences against analogous segments of the VP35 region of theEbola Sudan, Resteon, and Cote d'Ivoire strains. Thus, for example, SEQID NO:21 against Ebola VP35 For the Ebola virus is directed againstEbola viral sequence region 3136-3115, so exemplary target sequencesagainst the other three Ebola strains would target the same genomicsegment. For the Ebola virus, exemplary target sequences against theVP24 region include SEQ ID NOS:34-41 for the Ebola Zaire strain andanalogous targeting sequences against analogous segments of the VP35region of the Ebola Sudan, Resteon, and Cote d'Ivoire strains.

For the Marburg virus, an exemplary targeting sequence against the VP35region is SEQ ID NOS:47 and 48, and against the VP24 region, SEQ IDNO:57.

Additional targets include the terminal 25 base pair region of the viralmRNA transcripts as represented by the sequences complementary to theSEQ ID NOs:42 and 43. These targets are preferred because of their highdegree of sequence conservation across individual filovirus isolates.

The Ebola virus RNA sequences (Zaire Ebola virus, Mayinga strain) can beobtained from GenBank Accession No. AF086833. The particular targetingsequences shown below were selected for specificity against the EbolaZaire virus strain. Corresponding sequences for Ebola Ivory Coast, EbolaSudan and Ebola Reston (GenBank Acc. No. AF522874) are readilydetermined from the known GenBank entries for these viruses. Preferablytargeting sequences are selected that give a maximum consensus among theviral strains, particularly the Zaire, Ivory Coast, and Sudan strains,or base mismatches that can be accommodated by ambiguous bases in theantisense sequence, according to well-known base pairing rules.

GenBank references for exemplary viral nucleic acid sequencesrepresenting filovirus genomic segments are listed in Table 3 below. Thenucleotide sequence numbers in Table 3 are derived from the GenBankreference for the positive-strand RNA of Ebola Zaire (AF086833) andMarburg virus (229337). It will be appreciated that these sequences areonly illustrative of other sequences in the Filoviridae family, as maybe available from available gene-sequence databases of literature orpatent resources (See e.g. world wide web ncbi.nlm.nih.gov). Thesequences in Table 3 below, identified as SEQ ID NOS: 1-14, are alsolisted in the Sequence Listing table at the end of the specification.

The target sequences in Table 3 represent the 3′ terminal 30 bases ofthe negative sense viral RNA or the 125 bases surrounding the AUG startcodons of the indicated filovirus genes. The sequences shown are thepositive-strand (i.e., antigenomic or mRNA) sequence in the 5′ to 3′orientation. It will be obvious that when the target is the minus-strandvRNA, as in the case of the Str Inh 1 target (SEQ ID NOS:15 and 44) thetargeted sequence is the complement of the sequence listed in Table 3.

Table 3 lists the targets for exemplary Ebola viral genes VP35, VP24,VP30, VP40, L and NP. The proteins represent six of the seven proteinsencoded by Ebola. The target sequences for the AUG start codons of thesix genes are represented as SEQ ID NOS:1-6. The corresponding set oftarget sequences for Marburg virus are shown as SEQ ID NOS:8-13. The 3′terminal sequence of the minus-strand viral RNA (SEQ ID NOS:7 and 14)can also be targeted. The sequences shown in Table 3 for the 3′ terminalminus-strand targets (SEQ ID NOS:7 and 14) are the minus-strandsequences in a 5′-3′ orientation for Ebola and Marburg viruses,respectively.

TABLE 3 Exemplary Filovirus Nucleic Acid Target Sequences Gen NucleotideSEQ ID Name Bank No. Region Sequence (5′ to 3′) NO VP35-AUG AF0868333029-3153 AAUGAUGAAGAUUAAAACCUUCA 1 UCAUCCUUACGUCAAUUGAAUUCUCUAGCACUCGAAGCUUAUUGUC UUCAAUGUAAAAGAAAAGCUGGU CUAACAAGAUGACAACUAGAACAAAGGGCAGGG VP24-AUG AF086833 10245-10369 CGUUCCAACAAUCGAGCGCAAGG 2UUUCAAGGUUGAACUGAGAGUGU CUAGACAACAAAAUAUUGAUACU CCAGACACCAAGCAAGACCUGAGAAAAAACCAUGGCUAAAGCUACG GGACGAUACA VP30-AUG AF086833 8409-8533AGAUCUGCGAACCGGUAGAGUUU 3 AGUUGCAACCUAACACACAUAAAGCAUUGGUCAAAAAGUCAAUAGA AAUUUAAACAGUGAGUGGAGACA ACUUUUAAAUGGAAGCUUCAUAUGAGAGAGGAC VP40-AUG AF086833 4379-4503 AAACCAAAAGUGAUGAAGAUUAA 4GAAAAACCUACCUCGGCUGAGAG AGUGUUUUUUCAUUAACCUUCAU CUUGUAAACGUUGAGCAAAAUUGUUAAAAAUAUGAGGCGGGUUAUA UUGCCUACUG L-AUG AF086833 11481-11605GUAGAUUAAGAAAAAAGCCUGAG 5 GAAGAUUAAGAAAAACUGCUUAUUGGGUCUUUCCGUGUUUUAGAUG AAGCAGUUGAAAUUCUUCCUCUU GAUAUUAAAUGGCUACACAACAUACCCAAUAC NP-AUG AF086833 370-494 UGAACACUUAGGGGAUUGAAGAU 6UCAACAACCCUAAAGCUUGGGGU AAAACAUUGGAAAUAGUUAAAAG ACAAAUUGCUCGGAAUCACAAAAUUCCGAGUAUGGAUUCUCGUCCU CAGAAAAUCU Str. AF086833 30-1 UAAAAAUUCUUCUUUCUUUUUGU 7 Ihn 1(-) GUGUCCG VP35-AUG Z29337 2844-2968CUAAAAAUCGAAGAAUAUUAAAG 8 GUUUUCUUUAAUAUUCAGAAAAGGUUUUUUAUUCUCUUCUUUCUUU UUGCAAACAUAUUGAAAUAAUAA UUUUCACAAUGUGGGACUCAUCAUAUAUGCAAC VP24-AUG Z29337 10105-10229 UUCAUUCAAACACCCCAAAUUUU 9CAAUCAUACACAUAAUAACCAUU UUAGUAGCGUUACCUUUCAAUAC AAUCUAGGUGAUUGUGAAAAGACUUCCAAACAUGGCAGAAUUAUCA ACGCGUUACA VP30-AUG Z29337 8767-8891GAAGAACAUUAAGUGUUCUUUGU 10 UAGAAUUAUUCAUCCAAGUUGUUUUGAGUAUACUCGCUUCAAUACA ACUUCCCUUCAUAUUUGAUUCAA GAUUUAAAAUGCAACAACCCCGUGGAAGGAGU VP40-AUG Z29337 4467-4591 UCCCAAUCUCAGCUUGUUGAAUU 11AAUUGUUACUUAAGUCAUUCUUU UUAAAAUUAAUUCACACAAGGUA GUUUGGGUUUAUAUCUAGAACAAAUUUUAAUAUGGCCAGUUCCAGC AAUUACAACA L-AUG Z29337 11379-11503UCAUUCUCUUCGAUACACGUUAU 12 AUCUUUAGCAAAGUAAUGAAAAUAGCCUUGUCAUGUUAGACGCCAG UUAUCCAUCUUAAGUGAAUCCUU UCUUCAAUAUGCAGCAUCCAACUCAAUAUCCUG NP-AUG Z29337   3-127 CACACAAAAACAAGAGAUGAUGA 13UUUUGUGUAUCAUAUAAAUAAAG AAGAAUAUUAACAUUGACAUUGA GACUUGUCAGUCUGUUAAUAUUCUUGAAAAGAUGGAUUUACAUAGC UUGUUAGAGU Str. Z29337 30-1 CAAAAUCAUCAUCUCUUGUUUUU 14 Ihn 1(-) GUGUGUC

Targeting sequences are designed to hybridize to a region of the targetsequence as listed in Table 3. Selected targeting sequences can be madeshorter, e.g., 12 bases, or longer, e.g., 40 bases, and include a smallnumber of mismatches, as long as the sequence is sufficientlycomplementary to allow hybridization with the target, and forms witheither the virus positive-strand or minus-strand, a heteroduplex havinga T_(m) of 45° C. or greater.

More generally, the degree of complementarity between the target andtargeting sequence is sufficient to form a stable duplex. The region ofcomplementarity of the antisense oligomers with the target RNA sequencemay be as short as 8-11 bases, but is preferably 12-15 bases or more,e.g. 12-20 bases, or 12-25 bases. An antisense oligomer of about 14-15bases is generally long enough to have a unique complementary sequencein the viral genome. In addition, a minimum length of complementarybases may be required to achieve the requisite binding T_(m), asdiscussed below.

Oligomers as long as 40 bases may be suitable, where at least a minimumnumber of bases, e.g., 12 bases, are complementary to the targetsequence. In general, however, facilitated or active uptake in cells isoptimized at oligomer lengths less than about 30, preferably less than25. For PMO oligomers, described further below, an optimum balance ofbinding stability and uptake generally occurs at lengths of 15-22 bases.

The oligomer may be 100% complementary to the viral nucleic acid targetsequence, or it may include mismatches, e.g., to accommodate variants,as long as a heteroduplex formed between the oligomer and viral nucleicacid target sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Oligomer backbones which are less susceptible to cleavage bynucleases are discussed below. Mismatches, if present, are lessdestabilizing toward the end regions of the hybrid duplex than in themiddle. The number of mismatches allowed will depend on the length ofthe oligomer, the percentage of G:C base pairs in the duplex, and theposition of the mismatch(es) in the duplex, according to well understoodprinciples of duplex stability. Although such an antisense oligomer isnot necessarily 100% complementary to the viral nucleic acid targetsequence, it is effective to stably and specifically bind to the targetsequence, such that a biological activity of the nucleic acid target,e.g., expression of viral protein(s), is modulated.

The stability of the duplex formed between the oligomer and the targetsequence is a function of the binding T_(m) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(m) of an antisensecompound with respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques, Methods Enzymol. Vol. 154 pp. 94-107. Each antisenseoligomer should have a binding T_(m), with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 50° C. T_(m)'s in the range 60-80° C. or greaterare preferred. According to well known principles, the T_(m) of anoligomer compound, with respect to a complementary-based RNA hybrid, canbe increased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high T_(m) (50° C. or greater) at a length of 20bases or less are generally preferred over those requiring greater than20 bases for high T_(m) values.

The antisense activity of the oligomer may be enhanced by using amixture of uncharged and cationic phosphorodiamidate linkages as shownin FIGS. 2G and 2H. The total number of cationic linkages in theoligomer can vary from 1 to 10, and be interspersed throughout theoligomer. Preferably the number of charged linkages is at least 2 and nomore than half the total backbone linkages, e.g., between 2-8 positivelycharged linkages, and preferably each charged linkages is separatedalong the backbone by at least one, preferably at least two unchargedlinkages. The antisense activity of various oligomers can be measured invitro by fusing the oligomer target region to the 5′ end a reporter gene(e.g. firefly luciferase) and then measuring the inhibition oftranslation of the fusion gene mRNA transcripts in cell free translationassays. The inhibitory properties of oligomers containing a mixture ofuncharged and cationic linkages can be enhanced between, approximately,five to 100 fold in cell free translation assays.

Table 4 below shows exemplary targeting sequences, in a 5′-to-3′orientation, that target the Ebola Zaire virus (GenBank Acc. No.AF086833) according to the guidelines described above. The sequenceslisted provide a collection of targeting sequences from which additionaltargeting sequences may be selected, according to the general classrules discussed above. SEQ ID NOS:16-43 are antisense to the positivestrand (mRNA) of the virus whereas SEQ ID NO:15 is antisense to theminus strand viral RNA.

TABLE 4 Exemplary Antisense Oligomer Sequences Targeting Ebola ZaireTarget GenBank No. SEQ ID Name AF086833 Sequence 5′-3′ NO Str. Inh. 1    1-22 (-) CGGACACACAAAAAGAAAGAAG 15 strand L-AUG 11588-11567GTAGCCATTTAATATCAAGAGG 16 L′-AUG 11581-11600 TGGGTATGTTGTGTAGCCAT 17L-29-AUG 11552-11573 CAAGAGGAAGAATTTCAACTGC 18 L+4-AUG 11584-11604GTATTGGGTATGTTGTGTAGC 19 L+11-AUG 11591-11611 CGTCTGGGTATTGGGTATGTT 20VP35-AUG 3136-3115 GTTGTCATCTTGTTAGACCAGC 21 VP35′-AUG 3133-3152CCTGCCCTTTGTTCTAGTTG 22 VP35-22-AUG 3032-3053 GATGAAGGTTTTAATCTTCATC 23VP35-19-AUG 3115-3133 GTCATCTTGTAGACCAGC 24 VP35-16-AUG 3118-3133GTCATCTTGTTAGACC 25 VP35+2-AUG 3131-3152 CCTGCCCTTTGTTCTAGTTGTC 26NP-AUG 464-483 GGACGAGAATCCATACTCGG 27 NP+4-AUG 473-495CAGATTTTCTGAGGACGAGAATC 28 NP+11-AUG 480-499 CATCCAGATTTTCTGAGGAC 29NP+18-AUG 487-507 CTCGGCGCCATCCAGATTTTC 30 NP-19-AUG 451-472CATACTCGGAATTTTGTGATTC 31 VP40-AUG 4481-4498 GGCAATATAACCCGCCTC 32VP30-AUG 8494-8512 CCATTTAAAAGTTGTCTCC 33 VP24-AUG 10331-10349GCCATGGTTTTTTCTCAGG 34 VP24-28-AUG 10317-10336 CTCAGGTCTTGCTTGGTGTC 35VP24+4-AUG 10348-10369 TGTATCGTCCCGTAGCTTTAGC 36 VP24+10-AUG 10354-10372GATTGTATCGTCCCGTAGC 37 VP24+19-AUG 10361-10382 GGCGATATTAGATTGTATCGTC 38VP24-5′trm 10261-10280 TTCAACCTTGAAACCTTGCG 39 VP24(8+)-AUG 10331-10349GCCA+TGG+T+T+T+T+T+TC+TCAGG 40 VP24-5′trm(6+) 10261-10280+T+TCAACC+T+TGAAACC+T+TGCG 41 panVP35 3032-3053 GATGAAGGTTTTAATCTTCATC42 Scrv3 4390-4407 TTTTTCTTAATCTTCATC 43 8288-8305

In Table 4, above, SEQ ID NOs:40 and 41 are shown with cationic linkages(+) wherein X=(1-piperazino) as shown in FIG. 1B and FIG. 2H. Also inTable 4, above, SEQ ID NOs: 42 and 43 correspond to antisense oligomersthat target the 5′ terminal nucleotide region of the Ebola virus VP35mRNA (SEQ ID NO:42) and the 5′ terminal nucleotide region of both theEbola virus VP40 and VP30 mRNAs (SEQ ID NO:43).

Table 5 below shows exemplary targeting sequences, in a 5′-to-3′orientation, that target the Marburg virus (GenBank Acc. No. Z29337)according to the guidelines described above. The sequences listedprovide a collection of targeting sequences from which additionaltargeting sequences may be selected, according to the general classrules discussed above. SEQ ID NOS:45-58 are antisense to the positivestrand (mRNA) of the virus whereas SEQ ID NO:44 is antisense to theminus strand viral RNA.

TABLE 5 Exemplary Antisense Oligomer Sequences Targeting Marburg VirusTarget GenBank Name No. Z29337 Sequence 5′-3′ SEQ ID NO (-)3′term    1-21 (-) GACACACAAAAACAAGAGATG 44 L-AUG 11467-11485GCTGCATATTGAAGAAAGG 45 L+7-AUG 11485-11506 CATCAGGATATTGAGTTGGATG 46VP35-AUG 2932-2952 GTCCCACATTGTGAAAATTAT 47 VP35+7-AUG 2950-2971CTTGTTGCATATATGATGAGTC 48 NP-AUG  94-112 GTAAATCCATCTTTTCAAG 49 NP-6-AUG 97-120 CAAGCTATGTAAATCCATCTTTTC 50 NP+4-AUG 106-124CCTAACAAGCTATGTAAATC 51 NP-5′SL 68-88 TAACAGACTGACAAGTCTCAA 52 NP-5′UTR44-64 CAATGTTAATATTCTTCTTTA 53 NP-5′UTRb 36-56 ATATTCTTCTTTATTTATATGT 54VP30-AUG 8852-8873 GTTGCATTTTAAATCTTGAATC 55 VP35-5′UTR 2848-2867CCTTTAATATTCTTCGATTT 56 VP24+5-AUG 10209-10231 GTTGTAACGCGTTGATAATTCTG57 NP-stem loop 58-77 CAAGTCTCAATGTCAATGTT 58

III. Antisense Oligonucleotide Analog Compounds

A. Properties

As detailed above, the antisense oligonucleotide analog compound (theterm “antisense” indicates that the compound is targeted against eitherthe virus' positive-sense strand RNA or negative-sense or minus-strand)has a base sequence target region that includes one or more of thefollowing: 1) 125 bases surrounding the AUG start codons of viral mRNAand/or; 2) 30 bases at the 3′ terminus of the minus strand viral RNAand/or; 3) 25 bases at the 5′ terminus of viral mRNA transcripts. Inaddition, the oligomer is able to effectively target infecting viruses,when administered to a host cell, e.g. in an infected mammalian subject.This requirement is met when the oligomer compound (a) has the abilityto be actively taken up by mammalian cells, and (b) once taken up, forma duplex with the target RNA with a T_(m) greater than about 45° C.

As will be described below, the ability to be taken up by cells requiresthat the oligomer backbone be substantially uncharged, and, preferably,that the oligomer structure is recognized as a substrate for active orfacilitated transport across the cell membrane. The ability of theoligomer to form a stable duplex with the target RNA will also depend onthe oligomer backbone, as well as factors noted above, the length anddegree of complementarity of the antisense oligomer with respect to thetarget, the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Below are disclosed methods for testing any given, substantiallyuncharged backbone for its ability to meet these requirements.

B. Active or Facilitated Uptake by Cells

The antisense compound may be taken up by host cells by facilitated oractive transport across the host cell membrane if administered in free(non-complexed) form, or by an endocytotic mechanism if administered incomplexed form.

In addition to being substantially or fully uncharged, the antisenseagent is preferably a substrate for a membrane transporter system (i.e.a membrane protein or proteins) capable of facilitating transport oractively transporting the oligomer across the cell membrane. Thisfeature may be determined by one of a number of tests for oligomerinteraction or cell uptake, as follows.

A first test assesses binding at cell surface receptors, by examiningthe ability of an oligomer compound to displace or be displaced by aselected charged oligomer, e.g., a phosphorothioate oligomer, on a cellsurface. The cells are incubated with a given quantity of test oligomer,which is typically fluorescently labeled, at a final oligomerconcentration of between about 10-300 nM. Shortly thereafter, e.g.,10-30 minutes (before significant internalization of the test oligomercan occur), the displacing compound is added, in incrementallyincreasing concentrations. If the test compound is able to bind to acell surface receptor, the displacing compound will be observed todisplace the test compound. If the displacing compound is shown toproduce 50% displacement at a concentration of 10× the test compoundconcentration or less, the test compound is considered to bind at thesame recognition site for the cell transport system as the displacingcompound.

A second test measures cell transport, by examining the ability of thetest compound to transport a labeled reporter, e.g., a fluorescencereporter, into cells. The cells are incubated in the presence of labeledtest compound, added at a final concentration between about 10-300 nM.After incubation for 30-120 minutes, the cells are examined, e.g., bymicroscopy, for intracellular label. The presence of significantintracellular label is evidence that the test compound is transported byfacilitated or active transport.

The antisense compound may also be administered in complexed form, wherethe complexing agent is typically a polymer, e.g., a cationic lipid,polypeptide, or non-biological cationic polymer, having an oppositecharge to any net charge on the antisense compound. Methods of formingcomplexes, including bilayer complexes, between anionic oligonucleotidesand cationic lipid or other polymer components, are well known. Forexample, the liposomal composition Lipofectin® (Felgner, Gadek et al.1987), containing the cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. After administration, the complex is taken up by cells through anendocytotic mechanism, typically involving particle encapsulation inendosomal bodies.

The antisense compound may also be administered in conjugated form withan arginine-rich peptide linked covalently to the 5′ or 3′ end of theantisense oligomer. The peptide is typically 8-16 amino acids andconsists of a mixture of arginine, and other amino acids includingphenyalanine and cysteine. The use of arginine-rich peptide-PMOconjugates can be used to enhance cellular uptake of the antisenseoligomer (See, e.g. (Moulton, Nelson et al. 2004; Nelson, Stein et al.2005). Exemplary arginine-rich peptides are listed as SEQ ID NOs:61-66in the Sequence Listing.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., “Antisense oligonucleotides: A newtherapeutic principle,” Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747.

Alternatively, and according to another aspect of the invention, therequisite properties of oligomers with any given backbone can beconfirmed by a simple in vivo test, in which a labeled compound isadministered to an animal, and a body fluid sample, taken from theanimal several hours after the oligomer is administered, assayed for thepresence of heteroduplex with target RNA. This method is detailed insubsection D below.

C. Substantial Resistance to RNAseH

Two general mechanisms have been proposed to account for inhibition ofexpression by antisense oligonucleotides. (See e.g., (Agrawal, Mayrandet al. 1990; Bonham, Brown et al. 1995; Boudvillain, Guerin et al.1997). In the first, a heteroduplex formed between the oligonucleotideand the viral RNA acts as a substrate for RNaseH, leading to cleavage ofthe viral RNA. Oligonucleotides belonging, or proposed to belong, tothis class include phosphorothioates, phosphotriesters, andphosphodiesters (unmodified “natural” oligonucleotides). Such compoundsexpose the viral RNA in an oligomer:RNA duplex structure to hydrolysisby RNaseH, and therefore loss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and are believed to act bysterically blocking target RNA nucleocytoplasmic transport, splicing ortranslation. This class includes methylphosphonates (Toulme, Tinevez etal. 1996), morpholino oligonucleotides, peptide nucleic acids (PNA's),certain 2′-O-allyl or 2′-O-alkyl modified oligonucleotides (Bonham,Brown et al. 1995), and N3′→P5′ phosphoramidates (Ding, Grayaznov et al.1996; Gee, Robbins et al. 1998).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under a standard assay conditions, as described in Stein etal. After exposure to RNaseH, the presence or absence of intact duplexcan be monitored by gel electrophoresis or mass spectrometry.

D. In Vivo Uptake

In accordance with another aspect of the invention, there is provided asimple, rapid test for confirming that a given antisense oligomer typeprovides the required characteristics noted above, namely, high T_(m),ability to be actively taken up by the host cells, and substantialresistance to RNaseH. This method is based on the discovery that aproperly designed antisense compound will form a stable heteroduplexwith the complementary portion of the viral RNA target when administeredto a mammalian subject, and the heteroduplex subsequently appears in theurine (or other body fluid). Details of this method are also given inco-owned U.S. patent application Ser. No. 09/736,920, entitled“Non-Invasive Method for Detecting Target RNA” (Non-Invasive Method),the disclosure of which is incorporated herein by reference.

Briefly, a test oligomer containing a backbone to be evaluated, having abase sequence targeted against a known RNA, is injected into a mammaliansubject. The antisense oligomer may be directed against anyintracellular RNA, including a host RNA or the RNA of an infectingvirus. Several hours (typically 8-72) after administration, the urine isassayed for the presence of the antisense-RNA heteroduplex. Ifheteroduplex is detected, the backbone is suitable for use in theantisense oligomers of the present invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

When the antisense oligomer is complementary to a virus-specific regionof the viral genome (such as those regions of filovirus viral RNA ormRNA, as described above) the method can be used to detect the presenceof a given filovirus, or reduction in the amount of virus during atreatment method.

E. Exemplary Oligomer Backbones

Examples of nonionic linkages that may be used in oligonucleotideanalogs are shown in FIGS. 2A-2G. In these figures, B represents apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide,preferably selected from adenine, cytosine, guanine uracil. Suitablebackbone structures include carbonate (2A, R═O) and carbamate (2A,R═NH₂) linkages (Mertes and Coats 1969; Gait, Jones et al. 1974); alkylphosphonate and phosphotriester linkages (2B, R=alkyl or —O-alkyl)(Lesnikowski, Jaworska et al. 1990); amide linkages (2C) (Blommers,Pieles et al. 1994); sulfone and sulfonamide linkages (2D, R₁, R₂═CH₂);and a thioformacetyl linkage (2E) (Cross, Rice et al. 1997). The latteris reported to have enhanced duplex and triplex stability with respectto phosphorothioate antisense compounds (Cross, Rice et al. 1997). Alsoreported are the 3′-methylene-N-methylhydroxyamino compounds ofstructure 2F. Also shown is a cationic linkage in FIG. 2H wherein thenitrogen pendant to the phosphate atom in the linkage of FIG. 2G isreplaced with a 1-piperazino structure. The method for synthesizing the1-piperazino group linkages is described below with respect to FIG. 17.

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligonucleotides obeying Watson-Crick base-pairingrules, and mimic DNA in terms of base pair recognition (Egholm, Buchardtet al. 1993). The backbone of PNAs are formed by peptide bonds ratherthan phosphodiester bonds, making them well-suited for antisenseapplications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNAduplexes which exhibit greater than normal thermal stability. PNAs arenot recognized by nucleases or proteases.

A preferred oligomer structure employs morpholino-based subunits bearingbase-pairing moieties, joined by uncharged linkages, as described above.Especially preferred is a substantially unchargedphosphorodiamidate-linked morpholino oligomer, such as illustrated inFIGS. 1A-1D. Morpholino oligonucleotides, including antisense oligomers,are detailed, for example, in co-owned U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and5,506,337, all of which are expressly incorporated by reference herein.

Important properties of the morpholino-based subunits include: theability to be linked in a oligomeric form by stable, uncharged backbonelinkages; the ability to support a nucleotide base (e.g. adenine,cytosine, guanine or uracil) such that the polymer formed can hybridizewith a complementary-base target nucleic acid, including target RNA,with high T_(m), even with oligomers as short as 10-14 bases; theability of the oligomer to be actively transported into mammalian cells;and the ability of the oligomer:RNA heteroduplex to resist RNAsedegradation.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the β-morpholino subunit types shown in FIGS. 1A-1D,each linked by an uncharged, phosphorus-containing subunit linkage. FIG.1A shows a phosphorus-containing linkage which forms the five atomrepeating-unit backbone, where the morpholino rings are linked by a1-atom phosphoamide linkage. FIG. 1B shows a linkage which produces a6-atom repeating-unit backbone. In this structure, the atom Y linkingthe 5′ morpholino carbon to the phosphorus group may be sulfur,nitrogen, carbon or, preferably, oxygen. The X moiety pendant from thephosphorus may be fluorine, an alkyl or substituted alkyl, an alkoxy orsubstituted alkoxy, a thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures, such as morpholines or piperidines. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms. The Z moieties aresulfur or oxygen, and are preferably oxygen.

The linkages shown in FIGS. 1C and 1D are designed for 7-atomunit-length backbones. In Structure 1C, the X moiety is as in Structure1B, and the moiety Y may be methylene, sulfur, or, preferably, oxygen.In Structure 1D, the X and Y moieties are as in Structure 1B.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 1B,where X═NH₂ or N(CH₃)₂, Y═O, and Z=O.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged backbone linkages. One example of acationic charged phosphordiamidate linkage is shown in FIG. 2H. Thislinkage, in which the dimethylamino group shown in FIG. 2G is replacedby a 1-piperazino group as shown in FIG. 2G, can be substituted for anylinkage(s) in the oligomer. By including between two to eight suchcationic linkages, and more generally, at least two and no more thanabout half the total number of linkages, interspersed along the backboneof the otherwise uncharged oligomer, antisense activity can be enhancedwithout a significant loss of specificity. The charged linkages arepreferably separated in the backbone by at least 1 and preferably 2 ormore uncharged linkages.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above. Insome cases, it may be desirable to add additional chemical moieties tothe antisense compound, e.g. to enhance pharmacokinetics or tofacilitate capture or detection of the compound. Such a moiety may becovalently attached, typically to a terminus of the oligomer, accordingto standard synthetic methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 monomeric subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an antisense oligomer, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by a subject without undesirable sideeffects.

IV. Inhibition of Filovirus Replication

A. Inhibition in Vero Cells:

PMO antisense compounds and the control PMO (SEQ ID NO:35) wereinitially evaluated for cytotoxicity when incubated with Vero cells(FIG. 5) in the absence of virus. Only one PMO (0-1-63-308) was found toshow dose dependent cytotoxicity in the concentration range of 5 to 25μM.

Viral titer data were obtained 10 days post-infection of Vero cells. Allinhibitors evaluated to date reduce the viral titer, as measured byTCID₅₀, to some degree (Table 6 below) but the VP35-AUG inhibitor (SEQID NO:21) was the most inhibitory against Ebola virus. The inoculum wastaken from cells which received the treatment after infection (15 μM).No serum was added to media during pre-incubation with inhibitor orduring infection. After the infection the inoculum was removed andreplaced the medium containing 2% serum. The VP35-AUG PMO produced a 3log reduction in viral titer relative to the no treatment control group.The L-AUG PMO (SEQ ID NO:16) did not produce reduction in viral titer.The L gene is active later in the viral life cycle and the RNA becomeshighly bound by NP, VP30 and VP45 proteins so this target may beinaccessible to the L-AUG PMO used in this experiment.

TABLE 6 Viral Titer Reduction in Vero cells. Treatment (PMO) TCID₅₀TCID₅₀/ml Control (no treatment) −5.5 3.16 × 10⁶ VP35-scr (SEQ ID NO:60) −3.2 1.47 × 10⁴ Dscr (SEQ ID NO: 59) −3.8 6.81 × 10⁴ L-AUG (SEQ IDNO: 16) −4.2 1.47 × 10⁵ VP35-AUG (SEQ ID NO: 21) −2.5 3.16 × 10³

Vero cells were pretreated with different concentrations of the PMO(namely 0.1, 0.5, 1.0, 2.5, 5.0 and 10 μM), infected with EBOV for 1 hand the same concentration of PMO was added back afterwards. The 1 μMconcentration of VP35-AUG (SEQ ID NO:21) inhibitor reduced thecytopathic effect (CPE) significantly compared to 0.5 μM concentration.The reduction in CPE has been repeatedly observed in culture and anexample of these studies is seen in FIGS. 6A-6C for non-infected (FIG.6A), infected, no treatment (FIG. 6B), and infected and treated (FIG.6C).

B. Treatment in Infected Mice

These observations involved C57BL mice treated intraperitoneally (IP)with PMO at −24 and −4 hours prior to viral challenge at time 0. Eachtreatment group is composed of 10 male mice. The Ebola Zaire infectioninvolves 100 pfu injected IP and death is the endpoint observed betweendays 7 and 10 post infection. A summary of studies to date is providedin Table 7. The dose×2 indicates the dose given at the two times priorto viral infection. The VP35-AUGcon compound refers to the VP35-AUG PMOthat has been conjugated with the arginine-rich peptide R₉F₂C (SEQ IDNO:61).

TABLE 7 Summary of Treatment in Mice Group Dose Survivors/challengedSaline na 1/10 Str. Ihn 1 (SEQ ID NO: 15) 0.5 mg × 2 0/10 VP35-AUG (SEQID NO: 21) 0.5 mg × 2 6/10 Saline na 0/10 Str. Ihn 1 (SEQ ID NO: 15) 1.0mg × 2 2/10 VP35-AUG (SEQ ID NO: 21) 1.0 mg × 2 7/10 Scramble (SEQ IDNO: 60) 1.0 mg × 2 1/10 VP35-AUG + Str Inh 1 1.0 mg × 2 6/10 (SEQ IDNOs: 21 and 15) VP35-AUG-P003 + VP35-AUG 0.5 mg + 1.0 9/10 (SEQ ID NO:21 + 61 and 21) mg VP35-AUG-P003 (SEQ ID 0.5 mg × 2 9/10 NO: 21 + 61)

Mice that survived the viral challenge described in TABLE 7 wererechallenged with virus to determine the immunological consequence oftreatment. The results of the first studies are summarized in TABLE 8.

TABLE 8 Rechallenge Studies in Mice Group Earlier doseSurvivors/challenged Saline na  0/10 Str. Ihn 1, 0-1-63-412 0.5 mg × 21/2 VP35, 0-1-63-413 0.5 mg × 2 6/6 VP35, 0-1-63-413 1.0 mg × 2 7/7

All survivors from earlier Ebola challenge studies were evaluated inre-challenge studies 2 to 4 weeks after the initial viral exposure. TheMOI for the re-challenge was identical to the initial challenge. All ofthe re-challenged survivors from therapeutic treatment with the PMOtargeting VP35-AUG survived the re-challenge. These observations suggestviral replication was initiated in the viral challenge leading to arobust immune response, essentially a perfect vaccination. In accordancewith another aspect, the invention includes a method of vaccinating amammalian subject against Ebola virus by (i) pretreating the subjectwith antisense to Ebola virus, e.g., administering a VP35 antisense orcompound combination at one or two times prior to Ebola virus challenge,and (ii) challenging the individual with the virus, preferably in anattenuated form incapable of producing serious infection.

Similar treatment methods were aimed at determining the optimal lengthfor anti-Ebola PMO antisense, employing various length VP35 antisensePMO. As seen from the data below (Table 9) and the plot in FIG. 7, the16-mer is less effective than the 19-mer which is less effective thanthe 22-mer which is in the same order as the predicted Tm.

TABLE 9 Studies to Identify Optmal VP35-AUG Targeting Sequence Group AVINumber Survivors/challenged* Saline NA 1/10 VP35scr SEQ ID NO: 60 1/10VP35-16 SEQ ID NO: 25 3/10 VP35-19 SEQ ID NO: 24 5/10 VP35-22 SEQ ID NO:23 9/10 VP35′-AUG SEQ ID NO: 22 10/10; 9/10 *Observations as of day 9post challenge

Antisense compounds against six of the seven different genes expressedby Ebola were evaluated in the mouse model in a head-to-head experiment.(The GP gene was not included). As seen in Table 10, below, the mosteffective PMOs target VP24 and VP35 with L, VP40 and VP30 demonstratingless robust but significant activity in reducing mortality. The micetreated with VP40 died later than controls and those that survivedappeared to be less active. These data indicate differences in theefficacy for the different gene targets. As single antisense compounds,the antisense against VP35 and VP24 are preferred therapeutic agents.

TABLE 10 Comparison of Ebola Gene Targets Target SEQ ID NOSurvivors/challenged* NP-AUG 27 2/10 VP40-AUG 32 5/10 VP30-AUG 33 5/10VP24-AUG 34 10/10; 5/10 L′-AUG 17 6/10; 2/10 VP35-22 23 9/10 Scramble 600/10; 0/10 PBS NA 1/10; 0/10 *Dose 0.5 mg IP at −24 and −4 hours tochallenge, second survival numbers are from repeat experiment with freshvirus preparation.

In one embodiment, the antisense compound is administered in acomposition (or separately) in combination with one or more otherantisense compounds. One preferred combination is VP35-AUG (SEQ IDNO:21) plus VP24-AUG (SEQ ID NO:34); another is VP35-AUG, VP24-AUG andL-AUG (SEQ ID NOS:21, 34 and 16, respectively). As seen in Table 11below, and as plotted in FIG. 8, the dose response curves for acombination of the 3 compounds (VP35-AUG, VP24-AUG and L-AUG, each givenIP at 0.5 mg/dose) is not different from 5 different compounds(VP35-AUG, VP24-AUG, L-AUG, VP24-AUG and VP40-AUG). The dose of 0.5mg/mouse provides 100 percent survival from either combination. Further,these data indicate the EC₅₀ for combination therapy is between 10 and30 μg/mouse and that the EC₉₀ is approximately 50 μg/mouse.

TABLE 11 Combination Treatment for Ebola Survivors/ Group SEQ ID NOsChallenged PBS NA  0/10 NP-AUG, VP40-AUG, VP30-AUG, 27, 32-34 and 17 9/10 VP24-AUG, and L′-AUG NP-AUG, VP40-AUG, VP30-AUG, 27, 32-34, 17 and10/10 VP24-AUG, L′-AUG and VP35-AUG 21 VP35-AUG, VP24-AUG and L′-AUG 21,34 and 17 10/10 VP35-AUG, VP24-AUG, L′-AUG, 21, 34, 17, 32 and 10/10VP40-AUG and VP30-AUG 33 *Each agent in the combination administered 0.5mg via IP route.

The success with 100 percent survival from a single IP injection 24hours after Ebola infection (via IP route) indicates the antisensemechanism can suppress virus after viral replication has distributedthroughout the body and that these agents can be used as therapy forinfected individuals (Table 12 and FIG. 9). The comparison ofdose-response in the −24 and −4 hour regimen between VP35-AUG only andthe 3 agent combination is clear evidence of synergy. The combinationcould be more effective at 0.1× the dose than a single agent.

TABLE 12 Comparison of Dose Regimens Dose −24 and −4 −4 hours +24Treatment (mg) hours only hours VP35-AUG 0.5  9/10 (SEQ ID NO: 21) 0.05 4/10 VP35-AUG, VP24-AUG and 0.5 10/10 10/10  10/10  L′-AUG 0.05 10/104/10 5/10 (SEQ ID NOs: 21, 34 & 17) 0.005 0/10 4/10

V. Treatment Method

The antisense compounds detailed above are useful in inhibiting Ebolaviral infection in a mammalian subject, including human subjects.Accordingly, the method of the invention comprises, in one embodiment,contacting a cell infected with the virus with an antisense agenteffective to inhibit the replication of the virus. In one embodiment,the antisense agent is administered to a mammalian subject, e.g., humanor domestic animal, infected with a given virus, in a suitablepharmaceutical carrier. It is contemplated that the antisenseoligonucleotide arrests the growth of the RNA virus in the host. The RNAvirus may be decreased in number or eliminated with little or nodetrimental effect on the normal growth or development of the host.

A. Identification of the Infective Agent

The specific Ebola strain causing the infection can be determined bymethods known in the art, e.g. serological or cultural methods, or bymethods employing the antisense oligomers of the present invention.

Serological identification employs a viral sample or culture isolatedfrom a biological specimen, e.g., stool, urine, cerebrospinal fluid,blood, etc., of the subject. Immunoassay for the detection of virus isgenerally carried out by methods routinely employed by those of skill inthe art, e.g., ELISA or Western blot. In addition, monoclonal antibodiesspecific to particular viral strains or species are often commerciallyavailable.

Another method for identifying the Ebola viral strain employs one ormore antisense oligomers targeting specific viral strains. In thismethod, (a) the oligomer(s) are administered to the subject; (b) at aselected time after said administering, a body fluid sample is obtainedfrom the subject; and (c) the sample is assayed for the presence of anuclease-resistant heteroduplex comprising the antisense oligomer and acomplementary portion of the viral genome. Steps (a)-(c) are carried forat least one such oligomer, or as many as is necessary to identify thevirus or family of viruses. Oligomers can be administered and assayedsequentially or, more conveniently, concurrently. The viral strain isidentified based on the presence (or absence) of a heteroduplexcomprising the antisense oligomer and a complementary portion of theviral genome of the given known virus or family of viruses.

Preferably, a first group of oligomers, targeting broad families, isutilized first, followed by selected oligomers complementary to specificgenera and/or species and/or strains within the broad family/genusthereby identified. This second group of oligomers includes targetingsequences directed to specific genera and/or species and/or strainswithin a broad family/genus. Several different second oligomercollections, i.e. one for each broad virus family/genus tested in thefirst stage, are generally provided. Sequences are selected which are(i) specific for the individual genus/species/strains being tested and(ii) not found in humans.

B. Administration of the Antisense Oligomer

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. In accordance with the invention,routes of antisense oligomer delivery include, but are not limited to,various systemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery. The appropriate routemay be determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of a antisense oligomer in the treatment of a viralinfection of the skin is topical delivery, while delivery of a antisenseoligomer for the treatment of a viral respiratory infection is byinhalation. The oligomer may also be delivered directly to the site ofviral infection, or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically acceptable. Such a composition may include anyof a variety of standard pharmaceutically accepted carriers employed bythose of ordinary skill in the art. Examples include, but are notlimited to, saline, phosphate buffered saline (PBS), water, aqueousethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. The choice of suitable physiologicallyacceptable carrier will vary dependent upon the chosen mode ofadministration.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al, “Antisense oligonucleotides: A newtherapeutic principle,” Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747.

Sustained release compositions may also be used. These may includesemipermeable polymeric matrices in the form of shaped articles such asfilms or microcapsules.

The antisense compound is generally administered in an amount and mannereffective to result in a peak blood concentration of at least 200-400 nMantisense oligomer. Typically, one or more doses of antisense oligomerare administered, generally at regular intervals, for a period of aboutone to two weeks. Preferred doses for oral administration are from about5-1000 mg oligomer or oligomer cocktail per 70 kg individual. In somecases, doses of greater than 500 mg oligomer/patient may be necessary.For i.v., i.p or s.q. administration, preferred doses are from about100-1000 mg oligomer or oligomer cocktail per 70 kg body weight. Theantisense oligomer may be administered at regular intervals for a shorttime period, e.g., daily for two weeks or less. However, in some casesthe oligomer is administered intermittently over a longer period oftime. Administration may be followed by, or concurrent with,administration of an antibiotic or other therapeutic treatment. Thetreatment regimen may be adjusted (dose, frequency, route, etc.) asindicated, based on the results of immunoassays, other biochemical testsand physiological examination of the subject under treatment.

EXAMPLES Materials and Methods

Synthesis of PMOs. PMOs were designed with sequence homology near oroverlapping the AUG start site of Ebola virus VP35 (VP35′-AUG, SEQ IDNO:22), VP24 (VP24-AUG, SEQ ID NO:36), and L (L′-AUG, SEQ ID NO:17).Unrelated, scrambled PMOs (SEQ ID NOs:59 and 60) were used as a controlin all experiments. The PMOs were synthesized by AVI Biopharma, Inc.(Corvallis, Oreg.), as previously described (Summerton and Weller 1997).

A schematic of a synthetic pathway that can be used to make morpholinosubunits containing a (1-piperazino) phosphinylideneoxy linkage is shownin FIG. 17; further experimental detail for a representative synthesisis provided in Materials and Methods, below. As shown in the Figure,reaction of piperazine and trityl chloride gave trityl piperazine (1a),which was isolated as the succinate salt. Reaction with ethyltrifluoroacetate (1b) in the presence of a weak base (such asdiisopropylethylamine or DIEA) provided 1-trifluoroacetyl-4-tritylpiperazine (2), which was immediately reacted with HCl to provide thesalt (3) in good yield. Introduction of the dichlorophosphoryl moietywas performed with phosphorus oxychloride in toluene.

The acid chloride (4) is reacted with morpholino subunits (moN), whichmay be prepared as described in U.S. Pat. No. 5,185,444 or in Summertonand Weller, 1997 (cited above), to provide the activated subunits(5,6,7). Suitable protecting groups are used for the nucleoside bases,where necessary; for example, benzoyl for adenine and cytosine,isobutyryl for guanine, and pivaloylmethyl for inosine. The subunitscontaining the (1-piperazino) phosphinylideneoxy linkage can beincorporated into the existing PMO synthesis protocol, as described, forexample in Summerton and Weller (1997), without modification.

In vitro translation assay. The protein coding sequence for fireflyluciferase, without the initiator-Met codon ATG, was subcloned into themultiple cloning site of plasmid pCiNeo (Promega). Subsequently,complementary oligonucleotides for Ebola virus VP35 (−98 to +39 bases3020 to 3157), Ebola virus VP24 (−84 to +43 or bases 10261 to 10390),Ebola virus L (−80 to +49 or bases 11501 to 11632) were duplexed andsubcloned into Nhe 1 and Sal 1 sites. RNA was generated from the T7promoter with T7 Mega script (Ambion, Inc., Austin, Tex.). The in vitrotranslations were carried out by mixing different concentrations of PMOwith 6 nM RNA. A sigmoidal curve to determine the EC₅₀ values wasgenerated with the observed luciferase light emission (n=3 per PMOconcentration) and the PMO concentration.

Ebola virus infection of PMO-treated animals. C57Bl/6 mice, aged 8-10weeks of both sexes, were obtained from National Cancer Institute,Frederick Cancer Research and Development Center (Frederick, Md.). Micewere housed in microisolator cages and provided autoclaved water andchow ad libitum. Mice were challenged by intraperitoneal injection with˜1000 pfu of mouse-adapted Ebola virus diluted in phosphate bufferedsaline (PBS) (Bray, Davis et al. 1998). Mice were treated with acombination of 1 mg, 0.1, or 0.01 mg of each of the VP24-AUG, L′-AUG andVP35′-AUG PMOs (SEQ ID NOs:34, 17 and 22, respectively) or the scramblecontrol PMO (SEQ ID NO:60) either split between two equivalent doses at24 and 4 hours prior to Ebola virus challenge or a single dose 24 hoursafter challenge. C57Bl/6 mice were challenged intraperitoneally with1000 plaque-forming units of mouse-adapted Ebola virus (Bray, Davis etal. 1998). Hartley guinea pigs were treated intraperitoneally with 10 mgof each of the VP24-AUG, VP35′-AUG, and L′-AUG PMOs 24 hours before or24 or 96 hours after subcutaneous challenge with 1000 pfu of guinea-pigadapted Ebola virus (Connolly, Steele et al. 1999). Female rhesusmacaques of 3-4 kg in weight were challenged with 1000 pfu of Ebolavirus ('95 strain) (Jahrling, Geisbert et al. 1999) by intramuscularinjection following PMO treatment. The monkeys were treated from days −2through day 9 via a combination of parenteral routes as shown in FIG.10. The dose of the VP24-AUG PMO was 12.5-25 mg at each injection andthe dose of the VP35′-AUG and L′-AUG PMOs ranged from 12.5-100 mg perinjection.

Example 1 Antiviral Efficacy of Ebola Virus-Specific PMOs in Rodents

To determine the in vivo efficacy of the Ebola virus-specific PMOs, thesurvival of mice treated with 500 μg doses of the individual PMOs(VP24-AUG, L′-AUG and VP35′-AUG, SEQ ID NOs:34, 17 and 22, respectively)at 24 and 4 hours before challenge with 1000 plaque-forming units (pfu)of mouse-adapted Ebola virus was determined. The VP35′-AUG, VP24-AUG andL′-AUG PMOs exhibited a wide range of efficacy against lethal EBOVinfection and the VP35′-specific PMO provided nearly complete protection(FIG. 11A). Next, we performed a dose response experiment with the VP35PMO and found that reducing the dose of the PMO from 1,000 to 100 μgreduced the efficacy substantially (FIG. 11B). Hence, to further enhanceefficacy, we decided to use a combination of all three PMOs. Thiscombination of PMOs administered 24 and 4 h before lethal Ebola viruschallenge resulted in robust protection and showed substantialenhancement in protection afforded by the VP35 PMO alone, especially atlower doses (FIG. 11B). To determine the efficacy of the combination ofPMOs in a post-challenge treatment regimen, mice were injected with1,000 pfu of Ebola virus and were treated the next day with the PMOs(FIG. 11C). Mice that were given a single dose of 1,000 ug 24 h afterthe lethal challenge and survival was scored for 14 days. Again, Ebolavirus-infected mice were fully protected and lower doses showedsubstantial protection as compared to the control PMO. To determine theeffectiveness of the PMO treatment in Ebola virus-infected guinea pigs,the combination of PMOs was administered 24 hours before or 24 or 96hours after EBOV infection. Survival was greatly increased in guineapigs receiving the PMOs either 24 or 96 hours after infection, ascompared to untreated or pretreated guinea pigs as shown in FIG. 12.

Examination of tissues shortly following infection showed that treatmentof mice with the combination of Ebola virus-specific PMO slowed viralspread compared to mice treated with the scrambled PMO. Three days afterthe infection, multiple foci of infected cells were easily observed inthe spleens of the mice treated with the scrambled PMO (FIG. 11D). Incontrast, very few EBOV-infected cells could be found in the spleens ofthe anti-EBOV PMO-treated mice (FIG. 11E). Six days after viralinoculation, the infection was fulminant in the spleens of all animals(data not shown) and had spread to the livers of both mice treated withscrambled and combination PMOs (FIGS. 11F and 11G). However, the extentof the infection was limited in the combination PMO-treated mice, and,unlike the scrambled PMO-treated mice, EBOV antigen was not detectablewithin their hepatocytes, (FIGS. 11F and 11G). The observed pattern ofantigen staining within the tissues was corroborated by the viral titersas shown in FIG. 14.

To determine whether mice treated with the PMO therapeutics generatedimmune responses to Ebola virus, they were tested for Ebolavirus-specific cell-mediated and humoral immune responses. Four weeksafter infection, the mice demonstrated both CD4+ and CD8+ T cellresponses to multiple Ebola virus peptides, including NP and VP35 asshown in FIG. 14A. They also generated strong serum Ebola virus-specificantibody responses that were similar to the post-challenge antibodyresponses of mice protected by a therapeutic Ebola virus-like particlevaccine as shown in FIG. 14B (Warfield, Perkins et al. 2004). To studyif the generated immune responses were protective, PMO-treated mice wererechallenged with another dose of 1,000 pfu of Ebola virus four weeksafter surviving the initial challenge and these mice were completelyprotected from a second lethal Ebola virus infection as shown in FIG.14C.

Example 2 Antiviral Efficacy of Ebola Virus-Specific PMOs in Non-HumanPrimates

Based on the encouraging results both in vitro and in rodents, a trialin nonhuman primates was performed. Four rhesus monkeys were treatedwith PMO from two days prior to Ebola virus infection through day 9 ofthe infection. The naïve control monkey in this experiment received notreatment and succumbed to Ebola virus infection on day 10 as shown inFIG. 15A. Of 12 rhesus monkeys that have been infected in the inventors'laboratory with the same seed stock of virus, all died of Ebola virusbetween days 7 and 10 as shown in FIG. 15A. One of the PMO-treatedmonkeys succumbed to the infection on day 10. A second PMO-treatedmonkey cleared the EBOV infection from its circulation between days 9and 14, but was unable to recover from disease and died on day 16 asshown in FIGS. 15A and 15B. The two surviving monkeys had no symptoms ofdisease beyond mild depression until day 35, at which time they wereeuthanized. Incorporating historical controls, there were significantdifferences in survival curves between groups (p=0.0032). The meansurvival time for the treatment group was 14.3 days with a standarderror of 2.1 days. The mean survival time for the control group was 8.3days with a standard error of 0.2 days. The overall survival ratedemonstrated a significant p value of 0.0392, when compared tohistorical data.

There were early clinical signs or laboratory values that correlatedwith survival. The laboratory tests that most closely predicted survivalwere viral titers, platelet counts, and liver-associated enzymes in theblood. The monkeys that did not survive infection had detectable virusby day 5, in stark contrast to the PMO-treated monkeys that survived,which had little to no viremia on day 5 (FIG. 15B). As expected in ahemorrhagic disease, both the PMO-treated and naïve monkeys exhibitedthrombocytopenia. However, the PMO-treated monkeys that survived did nothave platelet counts far below 100,000 at any time, and their plateletcounts began to recover coincident with viral clearance (FIG. 15C).Similarly, all the monkeys experienced increases in theirliver-associated enzyme levels, including alkaline phosphatase. However,the levels in the surviving monkeys did not climb as high as those thatsuccumbed to the infection, and they returned to normal levels withinthe month after the EBOV infection (FIG. 15D). No correlation was foundbetween survival and multiple other hematological values, bodytemperature, serum cytokines, or fibrin degradation products. Since thesurviving PMO-treated monkeys had low to undetectable viremias followinginfection, we assessed the immune responses of the surviving monkeys. By28 days after Ebola virus challenge, the surviving rhesus monkeys hadhigh levels of both anti-EBOV antibodies and T cell responses, similarto the PMO-protected mice.

Example 3 Increased Antisense of Activity Using PMO with CationicLinkages

Two PMOs were synthesized using cationic linkages for a subset of theoligomer linkages as shown in Sequence Listing for SEQ ID NOs:40 and 41.These oligomers incorporated the cationic linkage (1-piperazinophosphoramidate) shown in FIG. 2H at the positions indicated with a “+”.These two PMOs target the EBOV VP24 mRNA. A cell free translation assaywas performed using the VP24:luciferase mRNA as the input RNA. PMO withand without cationic linkages were compared for their ability to inhibitluciferase expression and the results are shown in FIG. 16. Compared tothe uncharged PMO with the same base sequence, the PMOs with between 6and 8 cationic linkages demonstrated between 10 and 100-fold increasedantisense activity in this assay.

Based on the experiments performed in support of the invention asdescribed above in the Examples, efficacious anti-filovirus PMOs havebeen identified. The antiviral PMOs demonstrate favorable anti-Ebolaviral activity both in vitro and in vivo in both rodents and non-humanprimates. Together, the compounds and methods of the present inventionprovide a highly efficacious therapeutic treatment regimen for lethalEbola virus infections. PMOs have already been tested in clinical trialsand have appropriate pharmacokinetic and safety profiles for use inhumans (Arora and Iversen 2001). The results presented here havefar-reaching implications for the treatment of highly lethal Ebola virushemorrhagic fever, as well as diseases caused by other filovirusbiothreats including Marburg virus.

SEQUENCE LISTING TABLE SEQ ID AVI No. Name Target Sequences (5′-3′) NONA VP35-AUG AAUGAUGAAGAUUAAAACCUUCAUCAUCCUUACG 1UCAAUUGAAUUCUCUAGCACUCGAAGCUUAUUGU CUUCAAUGUAAAAGAAAAGCUGGUCUAACAAGAUGACAACUAGAACAAAGGGCAGGG NA VP24-AUG CGUUCCAACAAUCGAGCGCAAGGUUUCAAGGUUG 2AACUGAGAGUGUCUAGACAACAAAAUAUUGAUAC UCCAGACACCAAGCAAGACCUGAGAAAAAACCAUGGCUAAAGCUACGGGACGAUACA NA VP30-AUG AGAUCUGCGAACCGGUAGAGUUUAGUUGCAACCU 3AACACACAUAAAGCAUUGGUCAAAAAGUCAAUAG AAAUUUAAACAGUGAGUGGAGACAACUUUUAAAUGGAAGCUUCAUAUGAGAGAGGAC NA VP40-AUG AAACCAAAAGUGAUGAAGAUUAAGAAAAACCUAC 4CUCGGCUGAGAGAGUGUUUUUUCAUUAACCUUCA UCUUGUAAACGUUGAGCAAAAUUGUUAAAAAUAUGAGGCGGGUUAUAUUGCCUACUG NA L-AUG GUAGAUUAAGAAAAAAGCCUGAGGAAGAUUAAGA 5AAAACUGCUUAUUGGGUCUUUCCGUGUUUUAGAU GAAGCAGUUGAAAUUCUUCCUCUUGAUAUUAAAUGGCUACACAACAUACCCAAUAC NA NP-AUG UGAACACUUAGGGGAUUGAAGAUUCAACAACCCU 6AAAGCUUGGGGUAAAACAUUGGAAAUAGUUAAAA GACAAAUUGCUCGGAAUCACAAAAUUCCGAGUAUGGAUUCUCGUCCUCAGAAAAUCU NA Str. Ihn 1(-) UAAAAAUUCUUCUUUCUUUUUGUGUGUCCG7 NA VP35-AUG CUAAAAAUCGAAGAAUAUUAAAGGUUUUCUUAAU 8AUUCAGAAAAGGUUUUUUAUUCUCUUCUUUCUUU UUGCAAACAUAUUGAAAUAAUAAUUUUCACAAUGUGGGACUCAUCAUAUAUGCAAC NA VP24-AUG UUCAUUCAAACACCCCAAAUUUUCAAUCAUACAC 9AUAAUAACCAUUUUAGUAGCGUUACCUUUCAAUA CAAUCUAGGUGAUUGUGAAAAGACUUCCAAACAUGGCAGAAUUAUCAACGCGUUACA NA VP30-AUG GAAGAACAUUAAGUGUUCUUUGUUAGAAUUAUUC10 AUCCAAGUUGUUUUGAGUAUACUCGCUUCAAUAC AACUUCCCUUCAUAUUUGAUUCAAGAUUUAAAAUGCAACAACCCCGUGGAAGGAGU NA VP40-AUG UCCCAAUCUCAGCUUGUUGAAUUAAUUGUUACUU 11AAGUCAUUCUUUUUAAAAUUAAUUCACACAAGGU AGUUUGGGUUUAUAUCUAGAACAAAUUUUAAUAUGGCCAGUUCCAGCAAUUACAACA NA L-AUG UCAUUCUCUUCGAUACACGUUAUAUCUUUAGCAA 12AGUAAUGAAAAUAGCCUUGUCAUGUUAGACGCCA GUUAUCCAUCUUAAGUGAAUCCUUUCUUCAAUAUGCAGCAUCCAACUCAAUAUCCUG NA NP-AUG CACACAAAAACAAGAGAUGAUGAUUUUGUGUAUC 13AUAUAAAUAAAGAAGAAUAUUAACAUUGACAUUG AGACUUGUCAGUCUGUUAAUAUUCUUGAAAAGAUGGAUUUACAUAGCUUGUUAGAGU NA Str. Ihn 1(-) CAAAAUCAUCAUCUCUUGUUUUUGUGUGUC14 Ebola Virus Oligomer Targeting Sequences (5′-3′)  305 Str. Inh. 1CGGACACACAAAAAGAAAGAAG 15  309 L-AUG GTAGCCATTTAATATCAAGAGG 16  538L′-AUG TGGGTATGTTGTGTAGCCAT 17 1156 L-29-AUG CAAGAGGAAGAATTTCAACTGC 181157 L+4-AUG GTATTGGGTATGTTGTGTAGC 19 1158 L+11-AUGCGTCTGGGTATTGGGTATGTT 20  413 VP35-AUG GTTGTCATCTTGTTAGACCAGC 21  539VP35′-AUG CCTGCCCTTTGTTCTAGTTG 22  565 VP35-22-AUGGATGAAGGTTTTAATCTTCATC 23  540 VP35-19-AUG GTCATCTTGTAGACCAGC 24  541VP35-16-AUG GTCATCTTGTTAGACC 25 1151 VP35+2-AUG CCTGCCCTTTGTTCTAGTTGTC26  534 NP-AUG GGACGAGAATCCATACTCGG 27 1147 NP+4-AUGCAGATTTTCTGAGGACGAGAATC 28 1148 NP+11-AUG CATCCAGATTTTCTGAGGAC 29 1149NP+18-AUG CTCGGCGCCATCCAGATTTTC 30 1150 NP-19-AUG CATACTCGGAATTTTGTGATTC31  535 VP40-AUG GGCAATATAACCCGCCTC 32  536 VP30-AUG CCATTTAAAAGTTGTCTCC33  537 VP24-AUG GCCATGGTTTTTTCTCAGG 34 1152 VP24-28-AUGCTCAGGTCTTGCTTGGTGTC 35 1153 VP24+4-AUG TGTATCGTCCCGTAGCTTTAGC 36 1154VP24+10-AUG GATTGTATCGTCCCGTAGC 37 1155 VP24+19-AUGGGCGATATTAGATTGTATCGTC 38 0165 VP24-5′trm TTCAACCTTGAAACCTTGCG 39 0164VP24(8+)-AUG GCCA+TGG+T+T+T+T+T+TC+TCAGG 40 0166 VP24-5′trm(6+)+T+TCAACC+T+TGAAACC+T+TGCG 41 NA panVP35 GATGAAGGTTTTAATCTTCATC 42 NAScrv3 TTTTTCTTAATCTTCATC 43 Marburg Virus Oligomer Targeting Sequences(5′-3′) NA (-)3′term GACACACAAAAACAAGAGATG 44 NA L-AUGGCTGCATATTGAAGAAAGG 45 0177 L+7-AUG CATCAGGATATTGAGTTGGATG 46 NAVP35-AUG GTCCCACATTGTGAAAATTAT 47 0178 VP35+7-AUG CTTGTTGCATATATGATGAGTC48 NA NP-AUG GTAAATCCATCTTTTCAAG 49 0173 NP-6-AUGCAAGCTATGTAAATCCATCTTTTC 50 0174 NP+4-AUG CCTAACAAGCTATGTAAATC 51 0176NP-5′SL TAACAGACTGACAAGTCTCAA 52 NA NP-5′UTR CAATGTTAATATTCTTCTTTA 530175 NP-5′UTRb ATATTCTTCTTTATTTATATGT 54 NA VP30-AUGGTTGCATTTTAAATCTTGAATC 55 NA VP35-5′UTR CCTTTAATATTCTTCGATTT 56 0179VP24+5-AUG GTTGTAACGCGTTGATAATTCTG 57 NA NP-stem tooCAAGTCTCAATGTCAATGTT 58 Control Oligomers  183 DSscrAGTCTCGACTTGCTACCTCA 59  542 Scr TGTGCTTACTGTTATACTACTC 60 PeptideConjugates* NA R9F2C NH₂-RRRRRRRRRFFC-CO₂H 61 NA RXR4NH₂-RXRRXRRXRRXRXB-CO₂H 62 NA P008RX8 NH₂-RXRXRXRXRXRXRXRXB-CO₂H 63 NARX4 NH₂-RXRXRXRXB-CO₂H 64 NA RXR2 NH₂-RXRRXRXB-CO₂H 65 NA RXR3NH₂-RXRRXRRXRXB-CO₂H 66 GenBank Accession Number Name Target Regionsgi|10141003: EBOV Zaire GATGAAGATTAAAACCTTCATCATCCTTACGTCA 67 3032-3154Mayinga_VP35 ATTGAATTCTCTAGCACTCGAAGCTTATTGTCTTCAATGTAAAAGAAAAGCTGGTCTAACAAGATGAC AACTAGAACAAAGGGCAGGGG gi|33860540:EBOV Zaire GATGAAGATTAAAACCTTCATCATCCTTACGTCA 68 3032-3154 1995_VP35ATTGAATTCTCTAGCACTCGAAGCTTATTGTCCT CAATGTAAAAGAAAAGCTGGTCTAACAAGATGACAACCAGAACAAAGGGCAGGGG gi|52352969: EBOV SudanATGATGAAGATTAAAACCTTCATCATCCTTTAAA 69 3011-3163 Gulu_VP35AAGAGAGCTATTCTTTATCTGAATGTCCTTATTA ATGTCTAAGAGCTATTATTTTGTACCCTCTTAGCCTAGACACTGCCCAGCATATAAGCCATGCAGCAG GATAGGACTTATAGACA gi|22671623: EBOVReston GATGAAGATTAAAACCTTCATCGCCAGTAAATGA 70 3019-3180 PA_VP35TTATATTGTCTGTAGGCAGGTGTTTACTCCACCT TAAATTTGGAAATATCCTACCTTAGGACCATTGTCAAGAGGTGCATAGGCATTACCACCCTTGAGAAC ATGTACAATAATAAATTGAAGGTATG gi|450908:MARV GAAGAATATTAAAGGTTTTCTTTAATATTCAGAA 71 2853-2944 PoppVP35AAGGTTTTTTATTCTCTTCTTTCTTTTTGCAAAC ATATTGAAATAATAATTTTCACAA gi|10141003:EBOV Zaire GATGAAGATTAATGCGGAGGTCTGATAAGAATAA 72 9885-10370 Mayinga_VP24ACCTTATTATTCAGATTAGGCCCCAAGAGGCATT CTTCATCTCCTTTTAGCAAAGTACTATTTCAGGGTAGTCCAATTAGTGGCACGTCTTTTAGCTGTATA TCAGTCGCCCCTGAGATACGCCACAAAAGTGTCTCTAAGCTAAATTGGTCTGTACACATCCCATACAT TGTATTAGGGGCAATAATATCTAATTGAACTTAGCCGTTTAAAATTTAGTGCATAAATCTGGGCTAAC ACCACCAGGTCAACTCCATTGGCTGAAAAGAAGCTTACCTACAACGAACATCACTTTGAGCGCCCTCA CAATTAAAAAATAGGAACGTCGTTCCAACAATCGAGCGCAAGGTTTCAAGGTTGAACTGAGAGTGTCT AGACAACAAAATATTGATACTCCAGACACCAAGCAAGACCTGAGAAAAAACCATGGCTAAAGCTACGG GACGATACAA gi|33860540: EVOV ZaireGATGAAGATTAATGCGGAGGTCTGATAAGAATAA 73 9886-10371 1995_VP24ACCTTATTATTCAGATTAGGCCCCAAGAGGCATT CTTCATCTCCTTTTAGCAAAGTACTATTTCAGGGTAGTCCAATTAGTGACACGTCTCTTAGCTGTATA TCAGTCGCCCCTGAGATACGCCACAAAAGTGTCTCTAAGCTAAATTGGTCTGTACACATCTCATACAT TGTATTAGGGACAATAATATCTAATTGAACTTAGCCGTTTAAAATTTAGTGCATAAATCTGGGCTAAC TCCACCAGGTCAACTCCATTGGCTGAAAAGAAGCCTACCTACAACGAACATCACTTTGAGCGCCCTCA CATTTAAAAAATAGGAACGTCGTTCCAACAATCGAGCGCAAGGTTTCAAGGTTGAACTGAGAGTGTCT AGACAACAAAGTATCGATCCTCCAGACACCAAGCAAGACCTGAGAAAAAACCATGGCTAAAGCTACGG GACGATACAA gi|52352969: EBOV SudanATGATGAAAATTAATGAGAAGGTTCCAAGATTGA 74 9824-10324 Gulu_VP24CTTCAATCCAAACACCTTGCTCTGCCAATTTTCA TCTCCTTAAGATATATGATTTTGTTCCTGCGAGATAAGGTTATCAAATAGGGTGTGTATCTCTTTTAC ATATTTGGGCTCCCACTAGGCTAGGGTTTATAGTTAAGGAAGACTCATCACATTTTTTATTGAACTAG TCTACTCGCAGAATCCTACCGGGAATAGAAATTAGAACATTTGTGATACTTTGACTATAGGAAATAAT TTTCAACACTACCTGAGATCAGGTTATTCTTCCAACTTATTCTGCAAGTAATTGTTTAGCATCATAAC AACAACGTTATAATTTAAGAATCAAGTCTTGTAACAGAAATAAAGATAACAGAAAGAACCTTTATTAT ACGGGTCCATTAATTTTATAGGAGAAGCTCCTTTTACAAGCCTAAGATTCCATTAGAGATAACCAGAA TGGCTAAAGCCACAGGCCGGTACAAgi|22671623: EBOV Reston GATGAAGATTAATTGCGGAGGAATCAGGAATTCA 759832-10328 PA_VP24 ACTTTAGTTCCTTAAGGCCTCGTCCGAATCTTCATCAGTTCGTAAGTTCTTTTATAGAAGTCATTAGC TTCTAAGGTGATTATATTTTAGTATTAAATTTTGCTAATTGCTTGCTATAAAGTTGAAATGTCTAATG CTTAAATGAACACTTTTTTGAAGCTGACATACGAATACATCATATCATATGAAAACATCGCAATTAGA GCGTCCTTGAAGTCTGGCATTGACAGTCACCAGGCTGTTCTCAGTAGTCTGTCCTTGGAAGCTCTTGG GGAGACAAAAAGAGGTCCCAGAGAGTCCCAACAGGTTGGCATAAGGTCATTAACACCAGCATAGTCGG CTCGACCAAGACTGTAAGCGAGTCGATTTCAACTAAAAAGATTATTTCTTGTTGTTTAAACAAATTCC TTTTGTGTGAGACATCCTCAAGGCACAAGATGGCTAAAGCCACAGGCCGATACAA gi|450908: MARV GAAGAACATTAAGAAAAAGGATGTTCTTATTTTT76 9997-10230 Popp_VP24 CAACTAAACTTGCATATCCTTTGTTGATACCCTTGAGAGACAACTTTTGACACTAGATCACGGATCAA GCATATTTCATTCAAACACCCCAAATTTTCAATCATACACATAATAACCATTTTAGTAGCGTTACCTT TCAATACAATCTAGGTGATTGTGAAAAGACTTCCAAACATGGCAGAATTATCAACGCGTTACAA *“X” and “B” denote 6-aminohexanoic acidand beta-alanine, respectively. **SEQ ID NOs:40 and 41 incorporated thecationic linkage (1-piperazino phosphoramidate) as shown in FIG. 2H, atthe positions indicated with a “+”.

1. A method of treating Ebola virus infection in a subject, comprising:administering to a subject a therapeutically effective amount of anantisense oligomer of 12-40 morpholino subunits linked byphosphorous-containing intersubunit linkages which join a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,wherein at least 2 and no more than half of the total number ofintersubunit linkages are positively charged at physiological pH; andcomprising a targeting sequence which forms a heteroduplex with a targetsequence of the AUG start-site region of a positive-strand mRNA forEbola viral protein 24 (VP24); wherein the antisense oligomer inhibitsvirus production.
 2. The method of claim 1, wherein the targetingsequence forms a heteroduplex with the AUG start-site region defined bySEQ ID NO:2.
 3. The method of claim 2, wherein the targeting sequence iscomplementary to at least 12 contiguous bases of the sequence of SEQ IDNO:2.
 4. The method of claim 2, wherein the oligomer has 15-25 subunits.5. The method of claim 2, wherein the heteroduplex has a Tm ofdissociation of at least 45° C.
 6. The method of claim 1, wherein themorpholino subunits are joined by intersubunit linkages in accordancewith the structure:

wherein Y₁═O, Z═O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is selected from alkyl alkoxy; thioalkoxy; —NR₂,wherein each R is independently H or lower alkyl; or 1-piperazino. 7.The method of claim 1, wherein the Ebola virus is viral strain EbolaZaire.
 8. The method of claim 1, which further includes administering tothe subject a therapeutically effective amount of a second antisenseoligomer having the same structure recited in claim 1, but having atargeting sequence which forms a heteroduplex with a target sequence ofthe AUG start-site region of a positive-strand mRNA for Ebola viralprotein 35 (VP35).
 9. The method of claim 8, wherein the Ebola viralprotein 35 targeting sequence forms a heteroduplex with the AUGstart-site region defined by SEQ ID NO:
 1. 10. The method of claim 8,wherein the Ebola viral protein 35 targeting sequence is complementaryto at least 12 contiguous bases of the sequence of SEQ ID NO:1.
 11. Themethod of claim 1, wherein the targeting sequence comprises the sequenceof SEQ ID NO:34.
 12. The method of claim 11, wherein the targetingsequence consists of the sequence of SEQ ID NO:34.
 13. The method ofclaim 8, wherein the VP24 targeting sequence consists of the sequence ofSEQ ID NO:34, and the VP35 targeting sequence of the second oligomerconsists of the sequence of SEQ ID NO:22.
 14. The method of claim 13,wherein the antisense oligomers have between 2-8 piperazine-containingintersubunit linkages.
 15. The method of claim 13, wherein the oligomertargeted against VP24 and the oligomer targeted against VP35 areadministered sequentially.
 16. The method of claim 13, wherein theoligomer targeted against VP24 and the oligomer targeted against VP35are administered concurrently.